JPH05126832A - Immune analyzer and immunoassay method - Google Patents

Abstract

(57) [Summary] [Purpose] Enables simple and highly sensitive immunoassay of antigens and antibodies in specimens without special pretreatment. [Structure and effect] Matrix capable of infusion by capillary action ( 10) to the labeling reagent zone (16) and the specimen spotting zone (1
8), the detection zone (20), with a substrate solution tank at one end
A washing liquid tank (22, 24) was provided in the middle of (12). When the antigen is spotted on the specimen spotting zone, for example, the labeling reagent zone
The enzyme labeled antibody is contained in (16), and the immobilized antibody is contained in the detection zone (20). By removing the free labeled antibody from the detection zone with the washing liquid supplied from the washing liquid tank (22) before developing the substrate solution, the measurement noise source can be removed. The prozone phenomenon can be prevented by successively supplying the antigen or labeled antibody to the detection zone with the washing liquid supplied from the washing liquid tank (24) before developing the substrate solution. It is also possible to use two cleaning liquid tanks (26, 28) together.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay device for analyzing an antigen (or antibody) in a sample.

[Technical background] Analysis of biological components and drugs contained in blood, urine and the like is very important for diagnosis of pathological conditions and judgment of treatment progress and plays an important role in the field of clinical examination. It is preferable that such a clinical test can be easily carried out even in an examination room, and it is desirable that a self-diagnosis at home can be carried out easily and quickly without special equipment. If the analyte is an enzyme that has enzymatic activity (eg amylase) or a substrate that can be a substrate for a specific enzyme (eg urine glucose), use a filter paper on a strip impregnated with the reaction reagent as a sample (for urine or blood). The presence or absence of the test enzyme can also be measured by immersing the sample in its color. There is a so-called dipstick type that utilizes this. When the analyte is not directly involved in the color development reaction, the enzyme immunoassay (EIA method) can be used for the analysis. The EIA method is a method in which an antigen-antibody reaction is traced using an enzyme activity as a label and the amount of the antigen or antibody is quantified from this. The measurement system is divided into many methods depending on the measurement target, the labeling substance, the format of the antigen-antibody reaction at the time of measurement, and the presence or absence of B / F separation after the antigen-antibody reaction or the method thereof. In any case, it is necessary to prepare various reagents for the analysis, so the necessary reagents (antibody, enzyme-labeled antigen, enzyme substrate,
Various analytical elements provided with a coloring reagent, etc.) have been proposed.

[0003]

2. Description of the Related Art For example, Japanese Laid-Open Patent Publication No. 1-503439 (WO8)
8/08536) discloses an assay device which can be conveniently used without requiring a special pretreatment. As shown in FIG. 6, this is because a strip piece 50 of a water-absorbing material (for example, filter paper) capable of transporting a developing solution by a capillary action, a signal prevention zone 52, a sample receiving zone 54, a reagent zone 56, an indicator zone. While the strip 58 is provided, a developing solution tank 60 is provided at the end of the strip piece 50. The developing solution tank 58 contains a substrate for a labeling enzyme and a color-developing reagent, and the signal-preventing zone 52 is impregnated with a signal-preventing agent (for example, an enzyme inhibitor) that prevents coloring due to an enzymatic reaction. In the case where the sample is an antigen, the reagent zone 56 is impregnated with the enzyme-labeled antigen, and the indicator zone 58 contains the immobilized antibody. When the strip piece 50 is dipped in the developing tank after the sample solution is spotted on the sample receiving zone 54, the substrate solution moves the antigen in the sample receiving zone 54 and the enzyme-labeled antigen in the reagent zone 56 to the indicator zone 58.・ Diffuse.
As a result, the enzyme-labeled antigen and the sample antigen competitively react with the immobilized antibody in the indicator zone 58. The free antigen and the labeled antigen that have not bound to the immobilized antibody are washed out from the indicator zone 58 by the subsequently supplied substrate solution, and the labeled antigen that remains in inverse proportion to the amount of the test antigen remains in the indicator zone 58. The amount of the immobilized labeled antigen is quantified by the color reaction between the labeling enzyme and the substrate in the substrate solution. At this time, the reaction between the antigen-labeling enzyme and the substrate in the substrate solution during the development of the strip piece 50 is suppressed by the signal inhibitor supplied from the signal preventing zone 52. With the diffusion of the substrate solution, the labeled antigen, the signal inhibitor, and the test antigen impregnated in each zone are all washed out and moved to the diffusion tip of the substrate solution. Therefore, the reaction of the labeling enzyme is suppressed until the developed tip reaches the indicator zone 58. However, once the labeled antigen is immobilized by the immobilized antibody, the signal inhibitor is washed away with the substrate solution supplied subsequently together with the free labeled antigen, and thus the enzyme of the labeled antigen undergoes a color reaction at this point. be able to.

When the test antigen is a multivalent antigen, a labeled antibody can be used instead of the labeled antigen. In this case, the test antigen is detected in a form sandwiched between the immobilized antibody and the labeled antibody. (So-called sandwich method).

As described above, in this assay device, the presence or absence and the amount of the test antigen (or antibody) can be easily measured by measuring the coloration of the indicator zone. However, in this assay device, an enzyme inhibitor is required in order to suppress the reaction of the labeling enzyme which is moving by the substrate solution. In addition, since the antigenicity of an antigen, particularly a low-molecular-weight antigen, may be changed by enzyme labeling, a sandwich method in which an antibody is enzyme-labeled is used for this type of immunoassay device (element). Is generally more widely practiced. However, since this assay device moves the test antigen and the labeled antibody together to the indicator zone with one substrate solution and causes the binding reaction, the sandwich method applicable to this is 1
Only step method. But in the one-step method,
When the test antigen is present in excess, a large amount of the antigen-immobilized antibody conjugate and the antigen-labeled antibody conjugate are also produced, and these cannot bind to each other. That is, when an excess amount of antigen is present, a fixed antibody-antigen-labeled enzyme conjugate is not formed in proportion to the amount of the antigen, resulting in an apparently low measured value. In this way, when the antigen concentration reaches a high value exceeding a certain concentration (inhibition zone concentration: prozone), the stoichiometric antigen-antibody binding reaction does not occur, and the calibration curve shows biphasic. Such a prozone effect causes an erroneous determination as to whether the obtained measurement value corresponds to a high antigen concentration or a low antigen concentration. Therefore, it was necessary to determine the correct antigen concentration by diluting the test antigen in several steps and comparing the measured values. Diluting a test sample or assaying a plurality of diluted samples is cumbersome and increases test costs.

Further, there are the following problems. When the test substance is an antibody, an enzyme-labeled second antibody may be used. The second antibody is an antibody against an antibody (usually IgG), and is widely used because it is relatively easy to label with an enzyme and has versatility that can be used for measuring various antibodies. In addition to the test antibody, this second antibody also binds to IgG that is contaminated in the sample solution. Therefore, the test antibody is bound to the immobilized antigen in advance and then washed to obtain the contaminant Ig.
After removing G, the step of sequential reaction of contacting the test antigen with this enzyme-labeled second antibody must be taken. Therefore, the assay method using the second antibody could not be applied to the above-described assay device in which all the antigens / antibodies bind to each other in one step.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an immunoassay device which does not require the use of an enzyme inhibitor or the like and can easily perform highly sensitive measurement. To do. It is another object of the present invention to provide an immunoassay device that does not cause a biphasic reaction due to the prozone effect and does not cause a complicated dilution operation and an increase in test cost. Another object of the present invention is to provide an immunoassay device suitable for an assay method using a second antibody. Another object of the present invention is to provide an immunoassay method using this immunoassay device.

[0008]

The above object of the present invention is achieved by an immunoassay device in which at least one washing liquid tank for washing action and prevention of mutual interference of reagents is attached to a matrix which can be infused by a capillary action. Was done.

One of the preferred embodiments of the present invention is that an immunoassay device for analyzing a test antigen in a sample solution is provided with the following. (a) a matrix that can be infused by capillary action, (b) a sample spotting zone onto which the test antigen-containing sample liquid provided in the matrix is spotted, (c) the sample spotting zone at a position remote from the sample spot Provided in the matrix, a labeling reagent zone containing an antigen or antibody labeled with a labeling enzyme, (d) provided in the matrix at a position downstream of the sample spotting zone and the labeling reagent zone in the infusion direction, A detection zone containing an antibody immobilized therein, (e) a substrate attached to the matrix at a position upstream of the sample spotting zone and the labeling reagent zone in the infusion direction, and containing a substrate of the labeling enzyme Solution tank, (f) A washing solution tank attached to the matrix at a position upstream of the labeling reagent zone in the infusion direction and downstream of the substrate solution tank.

In this embodiment, a washing liquid tank is provided on the upstream side of the labeling reagent zone, and the washing liquid in the washing liquid tank is supplied to the matrix prior to the development and movement of the substrate solution from the substrate solution tank, and the test antigen and the labeled antigen are labeled. (Or labeled antibody) is infused / moved to the detection zone for competitive reaction, and free labeled antigen (or labeled antibody) not bound to the immobilized antibody is washed away. Thereafter, the substrate solution supplied from the substrate solution tank reacts with the labeling enzyme to generate a color or a detectable signal (fluorescence, luminescence, etc.). After the labeled antigen (or labeled antibody) is immobilized in the detection zone, it does not come into contact with this substrate solution until the substrate solution in the substrate solution tank is developed. Moreover, an enzyme inhibitor for suppressing the reaction of the labeled enzyme is not required. The position of the sample spotting zone is not particularly limited as long as the labeling reagent zone is located downstream of the washing liquid tank. When the test substance is an antibody, the detection zone can be similarly configured by including an immobilized antigen instead of the immobilized antibody.

Another preferred embodiment of the present invention is an immunoassay device for analyzing a test antigen in a sample solution, which comprises: (a) a matrix that can be infused by capillary action, (b) a sample spotting zone onto which the test antigen-containing sample liquid provided in the matrix is spotted, (c) the sample spotting zone at a position remote from the sample spot Provided in the matrix, a labeling reagent zone containing an antigen or antibody labeled with a labeling enzyme, (d) provided in the matrix at a position downstream of the sample spotting zone and the labeling reagent zone in the infusion direction, A detection zone containing the antibody immobilized therein, (e) a substrate solution tank containing a substrate for the labeling enzyme, which is attached to the matrix at a position upstream of the labeling reagent zone in the infusion direction, (f) A washing liquid tank attached to the matrix at a position between the labeling reagent zone and the sample spotting zone:

In this embodiment, a washing liquid tank is provided between the labeling reagent zone and the sample spotting zone. By supplying the washing solution from the washing solution tank to the matrix prior to the development / movement of the substrate solution from the substrate solution tank, either the test antigen or the labeled antigen (or labeled antibody) is infused into the detection zone first. It is moved and allowed to bind to the immobilized antibody. Free test antigen or labeled antigen (or labeled antibody) that has not bound to the immobilized antibody is washed away with a washing solution. After that, the remaining labeled antigen (or labeled antibody) or the test antigen reaches the detection zone together with the substrate solution supplied from the substrate solution tank. Unreacted reagents (free antigen,
(Antibodies) have already been removed and the immune reaction progresses sequentially, so that even if the test antigen is in excess, it does not show biphasicity due to the prozone effect. When the test substance is an antibody, the detection zone can be similarly configured by including an immobilized antigen instead of the immobilized antibody. In this case, the labeled reagent zone may be impregnated with the enzyme-labeled second antibody.

The most preferable embodiment of the present invention is that the immunoassay device for analyzing a test antigen in a sample solution is provided with the following: (a) a matrix capable of infusion by capillary action, (b) a sample spotting zone onto which a sample liquid containing a test antigen provided on the matrix is spotted, (c) an upstream side in the infusion direction with respect to the sample spotting zone Is provided in the matrix at the position
A labeling reagent zone containing an antigen or antibody labeled with a labeling enzyme, (d) provided in the matrix at a position downstream of the specimen spotting zone in the infusion direction, and containing the antibody immobilized therein. Detection zone, (e) a substrate solution tank containing a substrate for the labeling enzyme, which is attached to the matrix at a position on the upstream side in the infusion direction with respect to the labeling reagent zone,
(f) a first washing liquid tank attached to the matrix at a position downstream of the labeling reagent zone in the infusion direction and upstream of the specimen spotting zone, (g) infusion direction of the labeling reagent zone A second cleaning liquid tank attached to the matrix at a position upstream and downstream from the substrate solution tank.

In this embodiment, first, the first washing liquid is supplied from the first washing liquid tank, and the test antigen is bound to the immobilized antibody in the detection zone. Next, the second washing liquid is supplied from the second washing liquid tank to move the labeled antigen or labeled antibody to the detection zone. When a labeled antigen is used, this labeled antigen binds to the immobilized antibody that did not bind to the test antigen.
When a labeled antibody is used, this labeled antibody binds in such a manner as to sandwich the test antigen bound to the immobilized antibody. In this way, the labeling enzyme of the labeled antigen (or labeled antibody) directly (or indirectly) bound to the immobilized antibody finally reacts with the substrate solution supplied from the substrate solution tank to develop color. Further, in this aspect, since each reagent is sequentially supplied and reacted, so-called prozone phenomenon does not occur. Further, since unreacted labeled antigen (or labeled antibody) is removed by the washing liquid supplied from the second washing liquid layer, an enzyme inhibitor is used to suppress the enzymatic reaction caused by the free labeled antigen (or labeled antibody). do not have to. In each of these aspects, the enzyme-labeled reagent zone, the sample spotting zone, and the detection zone are provided in advance in the matrix, but the regions for spotting each reagent or sample at the time of use are used as these zones as necessary. It may be one. Alternatively, the substrate solution may be supplied from the matrix end at the time of use without providing the substrate solution tank.

[0015]

Detailed Description of the Configuration of the Invention: Overall Configuration of the First Embodiment FIG. 1 shows a sectional view of the first embodiment of the immunoassay device of the present invention. In this figure, reference numeral 10 is a matrix, which is made of a water-absorbing material capable of infusing a developing solution by a capillary action. A preferable material is filter paper, and a filter paper formed of cellulose, glass fiber or the like may be used. The matrix 10 is formed in a strip shape, a substrate solution tank 12 is attached to one end in the length direction, and a water absorption promoting member 14 is adjacent to the other end. Between the substrate solution tank 12 and the absorption pad 14, the labeling reagent zone 16, the sample spotting zone 18, and the detection zone 2 are arranged so as to traverse the length direction thereof.
0 is sequentially provided. A washing liquid tank 22 is provided at a position between the substrate solution tank 12 and the labeling reagent zone 16.

Specimen spotting zone As the specimen spotting zone 18, a partial region of the matrix 10 is used as it is. Samples spotted on the sample spotting zone 18 include liquid components such as plasma, serum, urine, blood cell extract, whole blood, and whole blood extract. When the sample contains non-liquid components such as blood cells, a filter layer is provided on the sample spotting zone so that only the liquid component is impregnated inside the sample spotting zone during use. Good. Examples of the measurement target in the sample include the following antigens and antibodies. 1) Drugs: theophylline, phenytoin, valproic acid, etc. 2) Low molecular hormones: thyroxine, estrogen, elasteradiol, etc. 3) Cancer markers: carcinoembryonic antigen (CEA), α-fetoprotein (AFP), fecal hemoglobin 4) Viruses: human immunodeficiency virus (HIV), adult T-cell leukemia virus (ATLV), type A, type B and C
Hepatitis B virus, etc. 5) Polymeric hormones: thyroid stimulating hormone (TSH),
Insulin, etc. 6) Cytokine: interleukin 1 (IL-1),
IL-2, IL-6, etc. 7) Cell growth factor: epidermal growth factor (ECGF), platelet-derived growth factor (PDGF), etc. 8) Viral DNA, RNA, etc. 9) Inflammation-related antigen: C-reactive protein (CRP) and the like, 10) Antibodies against the above-mentioned antigens, and fragments of Fab, Fab ′, F (ab ′) _{2 and the} like having antigen recognition sites of these antibodies.

Labeling Reagent Zone The labeling reagent zone 16 is impregnated with an enzyme labeling reagent. When the measurement target in the sample is an antigen, an enzyme-labeled antigen or enzyme-labeled antibody is impregnated as the enzyme-labeling reagent. When the measurement target in the sample is an antibody, it is impregnated with an enzyme-labeled antigen, an enzyme-labeled antibody or an enzyme-labeled second antibody. When an antibody is used, this antibody may be an antibody obtained by a conventional method, but the sensitivity is further improved by using a monoclonal antibody. In addition, this antibody is F (ab ') ₂ , Fab', F
It may be a fragment such as ab. Any labeling enzyme may be used as long as it reacts with the substrate solution supplied from the substrate solution tank 12 to directly or indirectly generate a detectable signal (coloring, fluorescence, luminescence, etc.). Examples of these labeling enzymes include alkaline phosphatase, peroxidase, β-D-galactosidase and the like. The enzyme labeling method is the enzyme immunoassay (“Protein / Nucleic Acid / Enzyme” Supplement No. 31 (1987),
Known methods described in Eiji Ishikawa et al., Kyoritsu Shuppan) can be used.

Detection Zone The detection zone 20 contains an immobilized antibody when the measurement target is an antigen, and an immobilized antigen when the measurement target is an antibody. Immobilization of these antibodies and antigens may be carried out by directly binding to the matrix by covalent bonding or the like, but it may also be bound to an insoluble carrier and contained in the matrix. Examples of such an insoluble carrier include polystyrene latex, red blood cells of various animals, gelatin particles (Japanese Patent Publication No. Sho 63-29223), and glass fiber.
In any case, the antibody or antigen contained in the detection zone 20 is detected by the substrate solution supplied from the substrate solution tank 12 in the cleaning solution supplied from the cleaning solution tank 22.
It may be configured so as not to move from 0.

Substrate Solution Tank The substrate solution tank 12 contains a substrate solution containing a reaction substrate of a labeling enzyme of a labeled antibody or a labeled antigen. The substrate solution tank 12 may have a structure capable of supplying the substrate solution to the matrix 10 when it is used. For example, the bag may be made of a fragile material (eg, aluminum foil, polyethylene film, etc.), and the bag may be broken at the time of use so that the cleaning liquid therein is supplied to the matrix 10. The capacity of the substrate solution tank 12 may be such that the substrate solution can be sufficiently supplied to the detection zone 20 and the enzyme reaction in the detection zone can be sufficiently ensured. This amount is determined in consideration of the capacity of the cleaning liquid tank 22.

The reaction substrate in the substrate solution may be any as long as it produces a signal (coloring, fluorescence, luminescence, etc.) that can be detected directly or indirectly by the reaction with the labeling enzyme, and the coenzyme required for the enzyme reaction. Etc. are also included. In order to optically detect the product generated by the reaction with the labeling enzyme, a known detection reagent composition may be contained together. An appropriate reaction substrate can be selected from known reagents according to the type of labeling enzyme. For example: a) Chromogenic substrate: 3,3 ', 5,
5'-tetramethyl betidine (TMB), 2,2'-azinodi (3-ethylbenzthiazoline-6'-sulfonic acid (ABT
S), diaminobenzidine (DAB), 5-bromo-4-chloro-3-as a substrate for alkaline phosphatase
Indolyl phosphate (BCIP) b) Fluorescent substrate: 4 as a substrate for alkaline phosphatase
-Methylumbellyphenyl phosphate (4MU
P), as a substrate for β-D-galactosidase, 4-methylumbelliferyl β-D-galactoside (4MUG), c) as a luminescent substrate: as a substrate for alkaline phosphatase, 3-
(2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), as a substrate for β-D-galactosidase, 3- ( 2'-spiroadamantane) -4-methoxy-4- (3 "-β-D-galactopyranosyl) phenyl-1,2-dioxetane (AMPGP
D), luminol or isoluminol and hydrogen peroxide as a substrate for peroxidase.

The substrate solution may appropriately contain an enzyme activator, a coenzyme, a surfactant, a pH buffering agent and the like. Preferred buffers include acetate buffer, borate buffer, Tris-HCl buffer, diethanolamine buffer and the like.

Washing liquid tank In the washing liquid tank 22, the labeled antigen (or labeled antibody) is washed out from the labeling reagent zone 16 and the test antigen (or the tested antibody) to be measured is washed out from the assay spotting zone 18, and is placed in the detection zone 20. It contains a cleaning liquid to be moved. The cleaning liquid tank 22 may have a structure capable of supplying the substrate solution to the matrix 10 when it is used. For example, the bag may be made of a fragile material (eg, aluminum foil, polyethylene film, etc.), and the bag may be broken at the time of use so that the cleaning liquid therein is supplied to the matrix 10. It is composed of a bag made of a fragile material (for example, aluminum foil, polyethylene film, etc.), and the cleaning liquid therein is supplied to the matrix 10 by breaking the bag at the time of use. The capacity is such that the labeled antigen (or labeled antibody) from the labeling reagent zone 16 and the test antigen (or test antibody) to be measured from the assay spotting zone 18 are moved to the detection zone 20, respectively, and
A free test antigen that did not undergo a binding reaction in the detection zone 20,
The amount may be sufficient to wash away the labeled antigen (or the labeling dye).

The washing solution may be any solution that does not adversely affect the immune reaction, and for example, phosphate buffered saline (PBS) or acetate buffered saline can be used. If necessary, a surfactant may be added to effectively remove and wash unreacted antibodies / antigens and contaminant proteins in the sample. As a preferred surfactant, Tween 20,
Examples include Triton X-100, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and the like.

Water Absorption Promoting Member The water absorption promoting member 14 promotes smooth development and movement of the cleaning liquid supplied from the cleaning liquid tank 22 and the substrate solution subsequently supplied from the substrate solution tank 12 in the matrix 10. Used for. Unidirectional movement of liquid components within the matrix 10 (from left to right in FIG. 1)
In order to secure the water absorption promoting member 14,
A material having a higher water absorption or a material having a larger water retention capacity is preferable. For example, a sponge or a filter paper having a higher water retention capacity than the matrix 10 is laminated to form a water absorption promoting member 1
It can be 4. It is not necessary to provide such a water absorption promoting member 14 as long as the matrix 10 can be made sufficiently long and the water absorption capacity on the downstream side of the detection zone 20 can be secured. However, it is preferable to provide such a water absorption promoting member in order to shorten the development time of the liquid component and reduce the size of the entire apparatus.

In order to simplify the description of the method of use , a case will be described in which the measurement target is an antigen and the labeling reagent contained in the labeling reagent zone 16 is an enzyme-labeled antibody. In this case, the detection zone 20 contains immobilized antibody. After spotting the specimen on the specimen spotting zone 18, the washing liquid tank 22 is broken and the washing liquid is supplied to the matrix 10. If the protruding member 22a penetrating the matrix 10 is provided below the cleaning liquid tank 22, the cleaning liquid tank 22 can be pressed and broken to easily supply the cleaning liquid to the matrix 10. The washing liquid supplied to the matrix 10 is developed in the right direction in FIG. 1 by a capillary action, and the enzyme-labeled antibody is supplied from the labeling reagent zone 16 to the specimen spotting zone 1 at the tip of the development.
The test antigens are sequentially washed out from 8 and reach the detection zone 20. In the detection zone 20, the immobilized antibody reacts with the test antigen and the enzyme-labeled antibody, and the test antigen is immobilized in a form sandwiched between the immobilized antibody and the enzyme-labeled antibody (so-called one-step sandwich method). .. The free labeled antigen and the enzyme-labeled antibody that have not bound to the immobilized antibody are continuously supplied from then on to the water absorption promoting member 1 from the detection zone 20 by the washing liquid.
It is washed away in 4 directions. After the development of the washing solution is completed, the substrate solution tank 12 is pressed against the projecting member 12a penetrating the matrix 10 to break this, and the substrate solution is supplied to the matrix 10. The substrate solution spreads in the matrix 10 and reaches the detection zone 20. The substrate in the substrate solution reacts with the labeling enzyme of the labeled antibody bound to the immobilized antibody to produce an optically detectable signal, eg a dye. At this time, since the unreacted free labeled antibody has already been removed by the washing solution, this does not become a noise source.

The amount of the produced dye can be measured by a reflection light measuring system or a color difference meter, and the amount of the test antigen in the sample can be determined by the principle of the colorimetric method using a calibration curve prepared in advance. Further, the presence or absence of the test antigen may be qualitatively measured by visually confirming the presence or absence of the produced dye. If a color chart (color chart) corresponding to the antigen concentration is prepared, semi-quantitative measurement is possible even by visual discrimination. When a fluorescent substrate is used as the enzyme substrate, it is measured with a fluorometer.
When a luminescent substrate is used, the amount of luminescence can be measured with a photon counter or the like, but the instant film 20a shown by the phantom line in FIG. 1 is placed on the detection zone 20 in advance, and this film is used after the assay. The exposure dose of 20a may be measured.

Although the above description applies the sandwich method, a competitive reaction type assay is also possible by using an enzyme-labeled antigen instead of the enzyme-labeled antibody. In this case, the amount of dye produced in the detection zone 20 is inversely proportional to the amount of test antigen. Measurement target, labeling reagent zone 1
The combinations of the labeling reagents in 6 and the immobilizing reagents in the detection zone are summarized in Table 1 below.

[0028]

[Table 1] ────────────────────────────────── Measurement target Labeled reagent zone Detection zone Reaction format ─── ─────────────────────────────── 1) Antigen enzyme-labeled antigen-immobilized antibody competitive type 2) Antigen-enzyme-labeled antibody-immobilized Antibody sandwich type 3) Antibody enzyme labeled antibody immobilized antigen competitive type 4) Antibody enzyme labeled antigen immobilized antibody sandwich type 5) Antibody enzyme labeled secondary antibody immobilized antigen sandwich type 6) Antibody enzyme labeled antigen immobilized secondary antibody sandwich Mold ──────────────────────────────────

The second antibody used in Table 1 is an antibody against the antibody to be measured, and anti-IgG, anti-IgM, anti-IgE or the like is used depending on the Ig class of the antibody to be measured. This point also applies to the second antibody described in the description of other embodiments. Although the labeling reagent zone 16 is upstream of the sample spotting zone 18 in FIG. 1, this arrangement may be reversed. It is sufficient that at least the labeling reagent zone 16 is located on the downstream side of the cleaning liquid tank 22.

Second Embodiment In the second embodiment shown in FIG. 2, the washing liquid tank 24 is attached downstream of the labeling reagent zone 16 and upstream of the sample spotting zone. Since the members having the same configurations as those of the first embodiment described in FIG. 1 are denoted by the same reference numerals in FIG. 2, the description thereof will not be repeated. The configuration of the cleaning liquid tank 24 is also the same as that of the cleaning liquid tank 22 shown in FIG. 1, and only the installation location is different, and thus the description thereof will be omitted. However, the labeling reagent zone 16A is different from the first embodiment in that, in addition to the enzyme labeling reagent, an enzyme inhibitor that suppresses the reaction of this enzyme is also contained.

The method of use when the object to be measured is an antigen and the labeling reagent contained in the labeling reagent zone 16A is an enzyme-labeled antibody will be described. In this case, the detection zone 20 contains immobilized antibody. After the test antigen-containing sample liquid is spotted on the specimen spotting zone 18, the washing liquid tank 24 is pressed against the projecting member 24a provided below and broken to supply the washing liquid to the matrix 10. The washing liquid which is developed and moved in the right direction in FIG. 2 by the action of capillaries wash out the test antigen from the specimen spotting zone 18 and move it to the detection zone 20 at the tip of the development, and fix the test antigen on the detection zone 20. Conjugated antibody. If there is free test antigen that did not bind to the immobilized antibody due to an excess of test antigen,
After that, the free test antigen is washed away from the detection zone 20 and removed by the washing liquid continuously supplied.

After the development of the cleaning liquid is completed, the protrusion member 12a penetrating the matrix 10 through the substrate solution tank 12 this time.
To break the substrate solution by pressing it onto the matrix 10
Supply to. The substrate solution spreads in the matrix 10 to wash out the enzyme-labeled antibody from the labeling reagent zone 16A and move it to the detection zone 20. The enzyme-labeled antibody binds to the test antigen bound to the immobilized antibody in the detection zone 20,
The test antigen is immobilized by immobilizing it with the immobilized antibody.
That is, a two-step sandwich type reaction format is used. Unreacted labeled antibody and enzyme inhibitor are washed out and removed by the substrate solution continuously supplied thereafter.
The labeled enzyme immobilized by removing the enzyme inhibitor reacts with the substrate to generate a detectable signal such as a dye. In this embodiment, the binding reaction of the antigen, the immobilized antibody, and the enzyme-labeled antibody can be carried out sequentially to remove the free antigen that has not bound to the immobilized antibody in the first-step reaction. Even if there is an excessive amount, the prozone phenomenon does not occur. Therefore, analysis up to a higher concentration range becomes possible.

Although the above explanation applies the two-step sandwich method, an enzyme-labeled antigen can be used instead of the enzyme-labeled antibody. In this case, the larger the amount of test antigen, the smaller the amount of enzyme-labeled antigen that can bind to the immobilized antibody. Measurement target, labeling reagent zone 16A
The combinations of the labeling reagents and the immobilizing reagents in the detection zone 20 are shown in Table 2 below.

[0034]

[Table 2] ────────────────────────────────── Measurement target Labeled reagent zone Detection zone Reaction format ─── ─────────────────────────────── 1) Antigen enzyme-labeled antigen-immobilized antibody-2) Antigen enzyme-labeled antibody-immobilized antibody Sandwich type 3) Antibody enzyme-labeled antibody-immobilized antigen-4) Antibody enzyme-labeled antigen-immobilized antibody sandwich type 5) Antibody-enzyme-labeled second antibody-immobilized antigen sandwich type 6) Antibody enzyme-labeled antigen-immobilized second antibody-capture type ──────────────────────────────────

Third Embodiment In the third embodiment shown in FIG. 3, two cleaning liquid tanks are provided. That is, as in the first embodiment, the substrate solution tank 12 is attached to the most upstream side of the matrix 10 and the absorption promoting member 14 is attached to the most downstream side thereof, and in the meantime, the matrix 10 is provided with the labeling reagent zone 16 and the sample from the upstream side. A spotting zone 18 and a detection zone 20 are sequentially provided. These configurations are exactly the same as in the first embodiment. Then, the first washing liquid tank 26 is provided between the labeling reagent zone 16 and the sample spotting zone 18, and the second washing liquid tank 28 is provided between the substrate solution layer 12 and the labeling reagent zone 16. .. The configurations of these two cleaning liquid tanks 26 and 28 are similar to those of the cleaning liquid tank 22 of the first embodiment. It is also the same that the protruding members 26a and 28a for breaking the tanks are provided below each tank.

The method of use when the object to be measured is an antigen and the labeling reagent contained in the labeling reagent zone 16 is an enzyme-labeled antibody will be described. In this case, the detection zone 20 contains immobilized antibody. After spotting the test antigen-containing sample liquid on the specimen spotting zone 18, the first washing liquid tank 26 is broken and the washing liquid is supplied to the matrix 10. As a result, the test antigen in the sample spotting zone 18 is washed out, moves to the detection zone 20, and binds to the immobilized antibody. Excess antigen to be detected is detected by the washing solution continuously supplied to the detection zone 20.
Washed from and removed. After the development of the first washing liquid is completed, when the second washing liquid tank is broken this time and the second washing liquid is supplied to the matrix, the labeled antibody in the labeling reagent zone 16 is washed out and moves to the detection zone 20. This enzyme-labeled antibody binds to the test antigen bound to the immobilized antibody in the detection zone 20, and is immobilized by sandwiching the test antigen with the immobilized antibody. That is, a two-step sandwich type reaction format is used. The unreacted labeled antibody is washed out and removed from the detection zone by the second washing liquid continuously supplied thereafter. Finally, when the substrate solution tank 12 is broken and the substrate solution is supplied to the matrix 10, the substrate reaches the detection zone 20 and reacts with the labeling enzyme of the labeled antibody immobilized in the detection zone 20. As a result, a detectable signal such as a dye corresponding to the amount of the test antigen is generated. In this embodiment, since the binding reaction of the antigen, the immobilized antibody and the enzyme-labeled antibody is sequentially performed, the prozone phenomenon can be prevented and the measurement concentration range can be broadened.
Further, since the unreacted labeled antibody is removed by the cleaning liquid supplied from the second cleaning liquid tank 28, this does not become a noise source. Measurement target, labeling reagent zone 16A
Table 3 below shows a preferable combination of the labeling reagent in the above and the immobilizing reagent in the detection zone 20.

[0037]

[Table 3] ────────────────────────────────── Measurement target Labeled reagent zone Detection zone Reaction format ─── ─────────────────────────────── 1) Antigen enzyme-labeled antigen-immobilized antibody-2) Antigen enzyme-labeled antibody-immobilized antibody Sandwich type 3) Antibody enzyme-labeled antigen-immobilized antigen-4) Antibody enzyme-labeled second antibody-immobilized antigen Sandwich type 5) Antibody enzyme-labeled antigen-immobilized second antibody-capture type ────────────── ──────────────────────

In No. 4) of Table 3, the enzyme-labeled second antibody is used. Since this second antibody is an antibody against IgG or the like, when the antibody-containing sample to be measured is serum or plasma, it will bind to immunoglobulin in blood.
However, as in the case of the third embodiment, since the contaminating protein in the sample solution is removed in advance by the first washing solution, even if such a sandwich type assay using the second antibody is applied, the contaminating immunoglobulin is interfered with. There is no such thing. In this respect, No. 1 in Table 1 of the first embodiment. The combination of 5 is significantly different from the point that the test sample is limited to those containing no contaminating immunoglobulin. That is, according to the third embodiment, the sandwich method using the second antibody can be easily applied to analyze the amount of antibody in a blood sample such as serum, plasma, or whole blood.

[0039]

Example 1 An immunoassay device to which the one-step sandwich method using CRP as a test antigen was applied was prepared. The configuration is the same as in FIG. 27 from the bottom of a glass fiber filter paper (Wattman DD100) with a width of 1 cm and a length of 15 cm
At a position of mm, 40 μl of 1 μg / ml POD-labeled anti-human CRP mouse antibody solution (containing 75 mM ascorbic acid as an enzyme inhibitor) was spotted (this site serves as a labeling reagent zone). Similarly, at a position 45 mm from the bottom edge of the filter paper,
40 μl of a 1% suspension of anti-human CRP mouse antibody-bonded latex was spotted (this site is a detection zone; the anti-human CRP mouse antibody used here is the PO
It is an antibody that recognizes an epitope different from the D-labeled anti-human CRP mouse antibody). The filter paper was freeze-dried to form a labeling reagent zone 16 and a detection zone 20 in the filter paper matrix 10. 0.1% TMB (3,3 ', 5,5'-
Tetramethyl betidine), 0.05% H ₂ O ₂ solution 70
0 μl was put in a bag made of aluminum foil and placed at a position 5 mm from the lower end of the filter paper 10 to form a substrate solution tank 12. 150
An aluminum foil bag containing μl of PBS (10 mM phosphate buffered saline) was placed at a position 35 mm from the lower end of the filter paper 10, and this was used as a washing liquid tank 22. Filter paper matrix 1
Cellulose filter paper (width 5 mm, length 20 m at the other end of 0
m, thickness 2 mm) was laminated to obtain a water absorption promoting member 14.
As a comparative example of the immunoassay device of Example 1 produced in this manner, an apparatus excluding the cleaning liquid tank 22 from Example 1 was produced.

40 mm from the lower end of the filter paper matrix 10
The position was defined as the sample spotting zone 18, and 50 μl of a human CRP solution of known concentration was spotted. Then, the cleaning liquid tank 22
Was crushed with a finger and the cleaning liquid was absorbed in the filter paper matrix 10. Subsequently, the substrate solution tank 12 was smashed, the substrate solution was absorbed by the filter paper matrix 10 and developed by capillary action. After 10 minutes, the color of the detection zone 20 was measured with a colorimeter (CR221, manufactured by Minolta).
The same operation was performed for the comparative example. The results are shown in Fig. 4.

As shown in FIG. 4, in the comparative example in which the cleaning operation using the cleaning liquid tank 22 was not performed, the CRP concentration was 500 ng /
Biphasic with a peak of ml appeared and the prozone phenomenon was observed. On the other hand, in Example 1 in which the washing liquid tank 22 was provided and the washing operation was performed, such a prozone phenomenon was not observed, and good linearity was exhibited up to a CRP concentration of 5000 ng / ml at which the measured value reached a plateau. This means not only that the concentration range measurable by the analyzer of the present embodiment is wide, but also that the concentration is not erroneously calculated to be low even when the test antigen has an extremely high concentration.

[0042]

Example 2 Using CRP as a test antigen, an immunoassay device to which the two-step sandwich method was applied was prepared. Its configuration is similar to that of FIG. 1 μg / ml alkaline phosphatase-labeled anti-human CRP mouse antibody solution (50 mM Tris-HCl buffer; pH) was placed 27 mm from the lower end of a glass fiber filter paper (Whatman DD100) having a width of 1 cm and a length of 15 cm.
40 μl of 7.0) was spotted (this site becomes the labeling reagent zone). Similarly, at a position 45 mm from the bottom edge of the filter paper,
40 μl of a 1% suspension of anti-human CRP mouse antibody-bonded latex was spotted (this site serves as a detection zone; the anti-human CRP mouse antibody used here is the alkaline phosphatase-labeled anti-human CRP mouse antibody). Is an antibody that recognizes a different epitope). The filter paper was freeze-dried to form a labeling reagent zone 16 and a detection zone 20 in the filter paper matrix 10. 0.2% BC
IP (5-bromo-4-chloro-3-indolyl phosphoric acid)
700 μl of a solution containing 0.1% nitrotetrazolium blue was placed in a bag made of aluminum foil and placed at a position 5 mm from the lower end of the filter paper 10 to form a substrate solution tank 12. 1
Place an aluminum foil bag containing 50 μl of 10 mM PBS at a position 35 mm from the lower end of the filter paper 10,
Was used as the cleaning liquid tank 26. Also 100 μl of 10 mM PB
The aluminum foil bag containing S is 25m from the bottom edge of the filter paper 10.
It was placed at the position of m and used as the second cleaning liquid tank 28.
A water absorption promoting member 14 was attached to the other end of the filter paper matrix 10 in the same manner as in Example 1.

The sample spotting zone 18 was defined at a position 40 mm from the lower end of the filter paper matrix 10 of the immunoassay device of Example 2 thus prepared, and 50 μl of a human CRP solution of known concentration was spotted. Then, the first cleaning liquid tank 26 was smashed with a finger so that the cleaning liquid was absorbed in the filter paper matrix 10. Subsequently, the second cleaning liquid tank 28 is crushed and
The washing liquid of No. 1 was absorbed into the filter paper matrix 10. Then, the substrate solution tank 12 was smashed and the substrate solution was absorbed and spread in the filter paper matrix 10. After 10 minutes, the color of the detection zone 20 is measured by a color difference meter (CR22, manufactured by Minolta Co., Ltd.).
It was measured in 1). The same operation was performed for the comparative example. As shown in FIG. 5, the immunoanalyzer of Example 2 in which two washing liquid tanks were provided and the two-step sandwich method by sequential reaction was applied showed excellent linearity.

[0044]

Example 3 Adult T-cell leukemia virus (HTLV-
The antibody of 1) was used as a test antibody to prepare an immunoassay device to which a two-step sandwich method using a second antibody was applied. The structure is the same as that shown in FIG. 3, and the combinations of reagents in each zone are shown in Table 3. It corresponds to the combination of 5.

HTLV-1 antigen-bound latex was prepared as follows. 200 μl of 2.5% latex suspension
In addition, 10 μg / ml HTLV-1 antigen solution (10 mM
The mixture was mixed with 800 μl of acetate buffer: pH 6.0 and stirred at room temperature for 18 hours. The suspension was centrifuged (2,500 rpm,
5 minutes) to obtain latex pellets. This is 2m
1% BSA solution (50 mM phosphate buffer: pH 6.0)
Resuspended in, and centrifuged in the same manner. This washing operation was repeated 3 times and then suspended in 0.5 ml of the above BSA solution to obtain a 1% suspension of HTLV-1 antigen-bound latex.
40 μl of this HTLV-1 antigen-bound latex suspension
Was spotted at a position 75 mm from the lower end of a glass fiber filter paper (DD100 manufactured by Whatman Co., Ltd.) having a width of 1 cm and a length of 15 cm (this site becomes a detection zone). Similarly, at a position 27 mm from the lower end of the filter paper, 40 μl of a solution (50 mM Tris-HCl buffer; pH 7.0) of 100 ng / ml alkaline phosphatase-labeled anti-human IgG mouse antibody (second antibody) was spotted. It becomes the reagent zone). The filter paper is freeze-dried and the labeling reagent zone 1 is added to the filter paper matrix 10.
6 and the detection zone 20 were formed. 0.2% AMPPD
(3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane) disodium salt solution (900 μl) was placed in an aluminum foil bag,
Place the filter paper 10 at a position 5 mm from the lower end and put it in the substrate solution tank 1
It was set to 2. An aluminum foil bag containing 150 μl of 10 mM PBS was placed at a position 50 mm from the lower end of the filter paper 10, and this was used as the first washing liquid tank 26. Again 100 μl
Aluminum foil bag containing 10mM PBS of
It was placed at a position 20 mm from the lower end, and this was used as the second cleaning liquid tank 28. A water absorption promoting member 14 was attached to the other end of the filter paper matrix 10 in the same manner as in Example 1. Comparative Example 3
As an example, an apparatus excluding the first cleaning liquid tank 26 from Example 1 was also manufactured.

A position of 65 mm from the lower end of the filter paper matrix 10 of the immunoanalyzer of Example 3 thus prepared is defined as the sample spotting zone 18, and 20 mM phosphoric acid (pH 7.
0), HTLV-1 antibody negative, weakly positive and strongly positive human serum samples diluted 50 times with a buffer containing 20% NGS (normal goat serum) were spotted at 50 μl each. Then, the first cleaning liquid tank 26 was smashed with a finger so that the cleaning liquid was absorbed in the filter paper matrix 10. Subsequently, the second washing liquid tank 28 was smashed and the second washing liquid was absorbed in the filter paper matrix 10. Then, the substrate solution tank 12 was smashed and the substrate solution was absorbed and spread in the filter paper matrix 10. After 10 minutes, Polaroid Instant Photo 615 photographic paper was placed on the detection zone 20 and exposed for 1 minute, and the emission image was measured with a colorimeter (CR22, manufactured by Minolta).
It was measured in 1). The same operation was performed for Comparative Example 3. The measurement was performed in duplicate. Table 4 shows the reflected light measurement values measured with a colorimeter.

[0047]

[Table 4] ───────────────────────────── HTLV-1 antibody sample Antireflection amount ───────── ──────── Example 3 Comparative Example 3 ───────────────────────────── Negative 1 29.1 26. 4 2 27.3 26.6 Weak positive 1 44.8 27.5 2 44.7 26.8 Strong positive 1 73.1 28.8 2 73.0 26.7 ─────────── ───────────────────

As shown in Table 4, by providing the first washing liquid tank 26, the HTLV-1 antibody could be detected. This result can be analyzed as follows. Comparative Example 3
Does not include the first cleaning liquid tank 26. Therefore, the test antibody (that is, the anti-HTLV-1 human antibody) comes into contact with the second antibody (that is, the anti-human IgG mouse antibody) that has moved by the washing solution supplied from the second washing solution tank. If the sample solution containing the test antibody does not contain human IgG other than the test antibody, no problem occurs. The test antibody binds to the enzyme-labeled second antibody and further moves to the detection zone 20, where it binds to the immobilized antigen. However, when the test antibody-containing sample liquid is a human blood sample such as serum, plasma, or whole blood, the second antibody is not limited to the test antibody and binds to human IgG. Upon passing through the sample spotting zone 18, it binds to blood IgG, which is much more present than the test antibody. As a result, the amount of the second antibody bound to the target test antibody is significantly reduced. On the other hand, in the immunoassay device of the third embodiment, first, only the test sample is moved to the detection zone by the cleaning liquid from the first cleaning liquid tank 26.
Only the test antibody binds to the immobilized antigen, and other contaminating IgG is washed away from the detection zone by the wash solution subsequently supplied. Therefore, after this, the enzyme-labeled antibody that has moved by the wash solution supplied from the second wash solution tank can be bound to the test antibody without being consumed by the contaminant IgG. As described above, it was shown that the immunoassay device of the present embodiment provided with two washing liquid tanks can be applied to the analysis method using the second antibody.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a first embodiment of an immunoassay device of the present invention.

FIG. 2 is a schematic diagram of a second embodiment of the immunoassay device of the present invention.

FIG. 3 is a schematic diagram of a third embodiment of the immunoassay system of the present invention.

FIG. 4 is a calibration curve showing the experimental results of Example 1 and Comparative Example 1.

5 is a calibration curve showing the experimental results of Example 2. FIG.

FIG. 6 is a schematic diagram of a conventional immunoassay device.

[Explanation of symbols]

 10 matrix 12 substrate solution tank 14 water absorption promoting member (absorption pad) 16 labeling reagent zone 18 sample spotting zone 20 detection zone 22, 24 cleaning solution tank 26 first cleaning solution tank 28 second cleaning solution tank

(72) Inventor Hideto Kaneko 4-6-7 Shimoochiai, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd. (72) Inventor Kazunori Koiwai 4-6-7 Shimochiai, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd. In the company

Claims (14)

[Claims] 1. An immunoassay device comprising a matrix capable of being infused by a capillary action with at least one washing liquid tank for washing action and prevention of mutual interference of reagents. 2. An apparatus in which an enzyme-labeled reagent zone, a sample spotting zone and a detection zone are provided in a matrix which has a substrate solution tank at one end and which can be infused by a capillary action, and which is upstream of the labeling reagent zone in the infusion direction. The immunoassay device according to claim 1, wherein the washing liquid tank is attached to the matrix at a position downstream of the substrate solution tank. 3. An apparatus in which an enzyme labeling reagent zone, a sample spotting zone and a detection zone are provided in a matrix capable of being infused by capillary action with a substrate solution tank attached at one end, wherein the labeling reagent zone and the sample spotting zone are provided. The immunoassay device according to claim 1, wherein the washing liquid tank is attached to the matrix at a position between and. 4. An apparatus in which an enzyme labeling reagent zone, a sample spotting zone, and a detection zone are provided in a matrix which has a substrate solution tank at one end and which can be infused by capillary action, and which is downstream of the labeling reagent zone in the infusion direction. And a first washing liquid tank attached to the matrix at a position upstream of the sample spotting zone, and at a position upstream of the labeling reagent zone in the infusion direction and downstream of the substrate solution tank. The immunoassay device according to claim 1, wherein a second washing liquid tank is attached to the matrix. 5. An immunoassay device for analyzing a test antigen in a sample liquid, comprising: (a) a matrix infusible by a capillary action; (b) a sample containing the test antigen provided on the matrix. A sample spotting zone in which a liquid is spotted, (c) a labeling reagent zone provided on the matrix at a position apart from the sample spotting zone, and containing an antigen or antibody labeled with a labeling enzyme, (d) A detection zone provided in the matrix at a position downstream of the sample spotting zone and the labeling reagent zone in the infusion direction, and containing the antibody immobilized therein, (e) the sample spotting zone and the labeling reagent A substrate solution tank attached to the matrix at a position upstream of the zone in the infusion direction, and containing a substrate for the labeling enzyme, (f) an upstream side of the labeling reagent zone in the direction of infusion and a downstream side of the substrate solution tank. The washing liquid tank which is attached to the matrix in position. 6. An immunoassay device for analyzing a test antibody in a sample liquid, comprising: (a) a matrix infusible by a capillary action; (b) a sample containing the test antibody provided in the matrix. A sample spotting zone on which a liquid is spotted, (c) containing any of an antigen, an antibody or a second antibody provided in the matrix at a position apart from the sample spotting zone and labeled with a labeling enzyme Labeling reagent zone, (d) the sample spotting zone and the detection zone provided in the matrix at a position downstream of the labeling reagent zone in the infusion direction, and containing the antigen immobilized therein, (e) A substrate solution tank that is attached to the matrix at a position upstream of the sample spotting zone and the labeling reagent zone in the infusion direction, and contains a substrate of the labeling reagent, (f) in the infusion direction upstream of the labeling reagent zone. The washing liquid tank which is attached to the matrix at a position downstream of the substrate solution reservoir. 7. A test antigen in a sample solution, comprising:
An immunoassay device for analyzing (a) a matrix that can be infused by capillary action, (b) before
The sample liquid containing the test antigen provided on the matrix is spotted.
(C) Separated from the sample spotting zone
It is provided on the matrix at different positions and labeled with a labeling enzyme.
Labeled reagent zone containing immobilized antigen or antibody, (d) before
Infusion method rather than the sample spotting zone and the labeling reagent zone
Provided in the matrix at a position on the downstream side, in which
A detection zone containing the antibody immobilized on (e) the label
At the position upstream of the sensory reagent zone in the infusion direction, the matri
Substrate attached to the substrate and containing the substrate for the labeling enzyme
Solution tank, (f) the labeling reagent zone and the sample spotting zone
Cleaning liquid attached to the matrix at a position between
Tank. 8. An immunoassay device for analyzing a test antibody in a sample solution, comprising: (a) a matrix injectable by capillary action; (b) a sample containing the test antibody provided in the matrix. A sample spotting zone on which a liquid is spotted, (c) containing any of an antigen, an antibody or a second antibody provided in the matrix at a position apart from the sample spotting zone and labeled with a labeling enzyme Labeling reagent zone, (d) the sample spotting zone and the detection zone provided in the matrix at a position downstream of the labeling reagent zone in the infusion direction, and containing the antigen immobilized therein, (e) Attached to the matrix at a position upstream of the labeling reagent zone in the infusion direction, a substrate solution tank containing a substrate of the labeling reagent, (f) at a position between the labeling reagent zone and the sample spotting zone. Mato The washing liquid tank which is attached to the box. 9. An immunoassay device for analyzing a test antigen in a sample liquid, comprising: (a) a matrix infusible by capillary action; (b) a sample containing the test antigen provided in the matrix. A sample spotting zone where a liquid is spotted, (c) provided in the matrix at a position on the upstream side in the infusion direction with respect to the sample spotting zone,
A labeling reagent zone containing an antigen or antibody labeled with a labeling enzyme, (d) provided in the matrix at a position downstream of the specimen spotting zone in the infusion direction, and containing the antibody immobilized therein. Detection zone, (e) a substrate solution tank containing a substrate for the labeling enzyme, which is attached to the matrix at a position on the upstream side in the infusion direction with respect to the labeling reagent zone,
(f) a first washing liquid tank attached to the matrix at a position downstream of the labeling reagent zone in the infusion direction and upstream of the specimen spotting zone, (g) infusion direction of the labeling reagent zone A second cleaning liquid tank attached to the matrix at a position upstream and downstream from the substrate solution tank. 10. An immunoassay device for analyzing a test antibody in a sample solution, comprising: (a) a matrix injectable by capillary action; (b) a sample containing the test antibody provided in the matrix. A sample spotting zone where a liquid is spotted, (c) provided in the matrix at a position on the upstream side in the infusion direction with respect to the sample spotting zone,
A labeling reagent zone containing either an antigen, an antibody or a second antibody labeled with a labeling enzyme, (d) provided in the matrix at a position downstream of the specimen spotting zone in the infusion direction, and A detection zone containing the immobilized antigen, (e) a substrate solution tank containing a substrate for the labeling reagent, which is attached to the matrix at a position upstream of the labeling reagent zone in the infusion direction, and (f) the label A first washing liquid tank attached to the matrix at a position downstream of the reagent zone in the infusion direction and upstream of the sample spotting zone, (g) upstream of the labeling reagent zone in the infusion direction, and A second cleaning liquid tank attached to the matrix at a position downstream of the substrate solution tank. 11. An immunoassay method characterized by using a matrix capable of being infused by a capillary action having at least one washing liquid tank for washing action and prevention of mutual interference of reagents. 12. A matrix solution tank is provided at one end of a matrix capable of being infused by a capillary action, and an enzyme labeling reagent zone, a sample spotting zone and a detection zone are respectively provided in this matrix, while the infusion solution is provided more than the labeling reagent zone. At a position upstream in the direction and downstream from the substrate solution tank, a sample solution is spotted on the sample spotting zone using an immunoassay device having a washing solution tank attached to the matrix, and then the washing solution is washed from the washing solution tank. Was supplied to the matrix, and then the substrate solution was supplied to the matrix from the substrate solution tank, and directly or indirectly immobilized in the detection zone in an amount depending on the amount of the analyte in the sample solution. An immunoassay method characterized by detecting a labeling reagent. 13. A matrix solution tank is attached to one end of a matrix capable of being infused by a capillary action, and an enzyme labeling reagent zone, a specimen spotting zone and a detection zone are respectively provided on the matrix, while the labeling reagent zone and the specimen are provided. Using an immunoanalyzer in which a washing liquid tank is attached to the matrix at a position between the spotting zone, a sample liquid is spotted on the specimen spotting zone, and then the washing liquid is supplied to the matrix from the washing liquid tank, and then, The substrate solution is supplied to the matrix from the substrate solution tank, and the labeled reagent immobilized in the detection zone is detected directly or indirectly in an amount depending on the amount of the analyte in the sample solution. Immunoassay method. 14. A substrate solution tank is attached to one end of a matrix capable of being infused by a capillary action, and an enzyme labeling reagent zone, a sample spotting zone and a detection zone are respectively provided in this matrix, and the infusion solution is provided more than the labeling reagent zone. A first washing liquid tank attached to the matrix at a position downstream in the direction and upstream from the sample spotting zone, and upstream in the infusion direction from the labeling reagent zone and downstream from the substrate solution tank. After depositing the sample solution on the specimen spotting zone using the immunoassay device in which the matrix and the second washing solution tank are attached to the matrix, the first washing solution is applied to the matrix from the first washing solution tank. Supply, and then the second cleaning liquid is supplied to the matrix from the second cleaning liquid tank, and then the substrate solution is supplied to the matrix from the substrate solution tank. , Immunoassay method and detecting directly or indirectly the labeled reagent immobilized in the detection zone in an amount that depends on the amount of the test object in the sample solution.