JP2003254975A - Immunity measuring method, and kit and device used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunity measuring method, with a wide measurement range and with few malfunctions, wherein an inspected substance in a specimen liquid is detected or measured, and a kit and a device used therefor. <P>SOLUTION: This method for detecting or measuring a specimen 13 in the specimen liquid includes processes A for causing cross-linkage to take place in the specimen 13 of two molecules or more, and B for causing a first specific bond substance 12 and a second specific bond substance 15 to react with the specimen 13, the substance 12 being fixed on a support body 11 and specifically bonded to the specimen 13 and the substance 15 becoming sensitized by a sensitizing substance and specifically bonded to the specimen 13; and C for detecting or measuring a complex including the substance 12, the substance 15, and the specimen 13, these being generated in the process B. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay method for detecting or measuring a test substance in a sample solution by utilizing an immune reaction, and a kit and a device used therefor.

[0002]

2. Description of the Related Art In recent years, various test methods have been used in clinical tests, and one of the test methods is a dry chemistry test method. The dry chemistry is a method in which a liquid sample is spotted on a reagent carried in a dry state on a support such as a film or a test paper, and the amount of a test substance in the sample solution is measured. As a device, a single layer type in which a reagent is supported on a single layer of support,
There is a multi-layer type in which a plurality of layers such as a development layer, a reaction layer, and a reagent layer are laminated. The features are that the reagent is already supported on the support, the reagent does not need to be adjusted, the reagent can be stored in a small space, and the amount of the sample liquid can be small.

As a typical test method by dry chemistry, there is a method utilizing a sandwich type immune reaction. In the sandwich-type immunoreaction, a first specific binding substance that specifically binds to a test substance is immobilized on a support that serves as an immunoreaction field, and a second specific binding substance that specifically binds to the test substance is added. Sensitize with a detectable sensitizer. When the sample solution and the second specific binding substance sensitized by the sensitizing substance are added to this support, if the test substance is present in the sample liquid, The sensitized second specific binding substance binds to the first specific binding substance immobilized on the support. Here, the test substance in the sample solution is detected or measured by detecting or measuring a signal based on the sensitizing substance immobilized on the support. Since the test substance reacts in such a manner that it is sandwiched between two specific binding substances, it is generally called a sandwich-type immune reaction.

[0004] As a device used in such an inspection method, there is an in-vitro diagnostic agent known as a pregnancy diagnostic agent, for example. FIG. 6 shows a schematic diagram of a sandwich type immune reaction in a typical pregnancy diagnostic drug. The pregnancy diagnostic agent detects human chorionic gonad hormone 73 (hereinafter abbreviated as hCG), which is a test substance, by a sandwich-type immune reaction.
The pregnancy diagnostic agent is sensitized with an antibody 72 against hCG immobilized on a support 71 made of a porous material such as a nitrocellulose membrane, and colored particles 75 such as gold colloid or blue latex that can be detected by coloration. An antibody 74 against hCG sensitized as a substance is included. As the reaction, as shown in FIG. 6, via hCG73,
A sandwich-type immune reaction occurs in which the immobilized antibody 72 and the sensitizing antibody 74 that have been immobilized bind to each other.

FIG. 7 shows the quantitative relationship between the reacting substances and the intensity of the detected signal in the sandwich type immune reaction.
And FIG. 8 will be described. FIG. 7 is a graph showing the relationship between the amount of the test substance and the detection sensitivity, and FIG. 8 is a schematic diagram showing the reaction state in three regions having different amounts of the test substance.

As shown in FIG. 7, the amount of the test substance can be roughly classified into three regions. As an example, description will be given using a pregnancy diagnostic agent. Here, the amount of immobilized antibody and the amount of sensitized antibody are constant. Urinary h added to pregnancy diagnostics
In the first region 81, in which the amount of CG, that is, the amount of sensitizing antibody compared to the amount of test substance is large, the intensity of the color reaction on the immobilized antibody 72, that is, the detection sensitivity depends on the amount of hCG, and The graph of detection sensitivity shows linearity (FIG. 8 (a)). h
When the amount of CG is increased, the amount of antibody immobilized on the support 71 is limited, so the graph of hCG amount and detection sensitivity shows no linearity, and all the immobilized antibodies 72 have h
When bound to the complex of CG73 and sensitizing antibody 74,
The detection sensitivity shows the highest value (second region 82, FIG. 8).
(B)).

[0007]

As a characteristic of the sandwich type immune reaction, further, in the third region 83 where the amount of hCG in urine is large, that is, in comparison with the amount of sensitizing antibody, urine added to the pregnancy diagnostic drug When the amount of medium hCG is large, there is a phenomenon in which the coloring intensity decreases. As shown in FIG. 8 (c), this phenomenon is caused by the immobilization antibody 72 being blocked by an excess of hCG73 that does not form a complex with the sensitizing antibody 74. Formed hCG7
3 results from the inability to react with the immobilized antibody 72.

In a device for the purpose of qualitative determination such as a pregnancy diagnostic drug, it suffices to be able to determine the presence or absence of a test substance, that is, the presence or absence of a signal on the device. The decrease in signal strength at is not a problem.

However, in the case of performing quantitative measurement, it is necessary to secure a measurement range required in a general clinical test, and if the detection sensitivity of the device falls within the measurement range, the calibration range cannot be secured, and The specifications are sufficient. Further, even when the detection sensitivity is ensured within the measurement range, two test substance concentrations are calculated for one measured value, which causes malfunction.

In view of the above-mentioned conventional problems, the present invention suppresses a decrease in signal intensity in an excessive amount range of a substance to be tested, so that the measurement range is wide and the malfunction does not occur easily. An object of the present invention is to provide an immunoassay method for detecting or measuring a substance, a kit and a device used therefor.

[0011]

In order to solve the above problems, the immunoassay method of the present invention is a method for detecting or measuring a test substance in a sample solution, which comprises two or more molecules of the test substance. A for cross-linking, a first specific binding substance that is fixed to a support and specifically binds to the test substance, and a second that is sensitized with a sensitizing substance and specifically binds to the test substance Step B of reacting the specific binding substance with the test substance, and detecting or measuring the sensitizing substance to generate the first specific binding substance and the second specific binding substance generated in step B. The method is characterized by including a step C of detecting or measuring a complex containing a substance and the test substance.

Further, the immunoassay kit of the present invention is a kit for detecting or measuring a test substance in a sample solution, wherein a first specific binding substance which specifically binds to the test substance is immobilized. And a second specific binding substance that is sensitized by the sensitizing substance and specifically binds to the test substance, and a cross-linking substance that cross-links two or more molecules of the test substance. And

The immunodevice of the present invention is a device for detecting or measuring a test substance in a sample liquid, which comprises a sample introducing section, a labeling section and a judging section, and the labeling section is A second specific binding substance that is sensitized by the preparation substance and that specifically binds to the test substance is carried in a state that can be eluted by the sample solution, and the determination unit specifically binds to the test substance. The first specific binding substance to be bound is fixed, and the sample introducing part, the labeling part and the judging part are moved so that the sample solution introduced into the sample introducing part moves to the judging part via the labeling part. It is arranged that the sample liquid contains a cross-linking substance that cross-links two or more molecules of the test substance in the path until the sample liquid reaches the determination unit.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The immunoassay method of the present invention is a method for detecting or measuring a test substance in a sample solution, which comprises a step A of crosslinking two or more molecules of the test substance, fixing to a support. The first specific binding substance that specifically binds to the test substance, and the second specific binding substance that is sensitized by the sensitizing substance and specifically binds to the test substance, And a complex containing the first specific binding substance, the second specific binding substance, and the test substance generated in step B by detecting or measuring the sensitizing substance. The method is characterized by including the step C of detecting or measuring the body.

By doing so, as shown in FIG. 1, in the excessive amount region of the test substance, an excess amount of the substance which does not form a complex with the second specific binding substance 15 sensitized by the sensitizing substance is formed. Even when the first specific binding substance 12 immobilized on the support 11 is blocked by the test substance 13, the test substance forming a complex with the second specific binding substance 15 sensitized by the sensitizing substance Substance 13 and first specific binding substance 12
Since the sensitizing substance is immobilized on the support 11 by cross-linking the test substance 13 bound to the test substance 13 via the cross-linking substance 14, it is possible to suppress a decrease in signal intensity.

The immunoassay method of the present invention can be used in any known method as long as it is a method of utilizing a sandwich type immunoreaction, for example, ELIZA method, immunochromatography method, immunoconcentration method, immunoblotting method. Etc.

Further, the immunoassay kit of the present invention is a kit for detecting or measuring a test substance in a sample solution, wherein a first specific binding substance which specifically binds to the test substance is immobilized. And a second specific binding substance that is sensitized by the sensitizing substance and specifically binds to the test substance, and a cross-linking substance that cross-links two or more molecules of the test substance. And

By using such a kit, the same effects as those of the immunoassay method of the present invention can be obtained. here,
For example, by using an enzyme as a sensitizing substance and an antibody against a test substance as a specific binding substance, it can be applied to an immunoassay kit using the ELISA method. An enzyme-sensitized antibody that specifically binds to a test substance sensitized by a sample solution containing the test substance and an enzyme to a support on which an antibody that specifically binds to the test substance is immobilized Is added, the test substance in the sample solution is captured by the immobilized antibody, and the captured test substance binds to the enzyme-sensitized antibody. The detection is performed by adding a substrate and causing an enzymatic reaction. The immunoassay kit of the present invention can be used, for example, in addition to the ELISA method.
It can be used in RIA (radioimmunoassay), CLIA (chemiluminescence immunoassay), ELIA (electrochemiluminescence) and the like.

The immune device of the present invention is a device for detecting or measuring a test substance in a sample solution, which comprises a sample introducing section, a labeling section and a judging section, and the labeling section is A second specific binding substance that is sensitized by the preparation substance and that specifically binds to the test substance is carried in a state that can be eluted by the sample solution, and the determination unit specifically binds to the test substance. The first specific binding substance to be bound is fixed, and the sample introducing part, the labeling part and the judging part are moved so that the sample solution introduced into the sample introducing part moves to the judging part via the labeling part. It is arranged that the sample liquid contains a cross-linking substance that cross-links two or more molecules of the test substance in the path until the sample liquid reaches the determination unit.

By using such a device,
The same effect as the immunoassay method of the present invention can be obtained. The immune device of the present invention can be used, for example, in an immunochromatography method, an immunoconcentration method, or the like.

The applications of the present invention include, for example, urine test, pregnancy test, water quality test, stool test, soil analysis, and food analysis. The sample solution may be either an aqueous solution or an organic solution, for example, body fluid, river water, seawater, groundwater,
Examples thereof include an aqueous solution in which soil or food is dissolved. Among these, examples of the body fluid include blood, plasma, serum, urine, saliva, sweat and tears.

Examples of test substances and specific binding substances in the present invention include antigens and antibodies. As an antigen,
Examples include bacteria, proteins and viruses.
Bacteria include erythrocytes, tubercle bacilli, and Escherichia coli, and examples of proteins include hCG, hemoglobin, hemoglobin A1c, myoglobin, and high density lipoprotein (HD
L), low-density lipoprotein (LDL), C-reactive protein (CRP), albumin, glycoalbumin, immunoglobulin, serum amyloid A or α-fetoprotein (AFP), etc., as HIV virus, or hepatitis C Examples include viruses. Examples of the antibody include anti-cell antibody, anti-protein antibody, anti-glycoprotein antibody, anti-enzyme antibody, anti-polysaccharide antibody, anti-bacterial antibody, anti-virus antibody and the like. The antibody is
It may be a monoclonal antibody or a polyclonal antibody.

Here, the test substance may be an antigen and an antibody against the antigen may be used as the specific binding substance, or an antigen may be used as the specific binding substance and the test substance may be an antibody against the antigen. For example, when the antigen is HIV virus, it is preferable that the antigen is used as the specific binding substance and the test substance is an antibody against the antigen.

Sensitizing substances include coloring substances, fluorescent substances,
Examples include phosphorescent substances, luminescent substances, redox substances, enzymes, nucleic acids, endoplasmic reticulum and the like. As the coloring substance, gold colloid, silver colloid, selenium colloid, colored latex, cyanine and azo, etc., as the fluorescent substance, aromatic compounds such as pyrene, those having a functional group substituted with an aromatic compound such as dansyl, Fluorescein, rhodamine, and coumarin, phosphorescent substances such as benzophenone, luminescent substances that emit light by a luminescent reaction between luciferin and ATP, and redox substances include:
Those that generate an electric current by a form such as a redox reaction between glucose and glucose oxidase, and vesicles include micelles and liposomes. Among these, gold colloid particles are more preferable.

Any support can be used as long as it can immobilize the first specific binding substance, and examples thereof include porous materials such as nitrocellulose membrane, cellulose acetate membrane, glass filter paper, polystyrene, and polyvinyl. , Polycarbonate, polypropylene, silicon-based, glass-based, dextran-based substrates and the like.

The material for the sample introducing part, the labeling part and the judging part may be any as long as the sample solution can be developed at an appropriate speed by the capillary phenomenon, for example, nitrocellulose membrane, cellulose acetate membrane, glass filter paper. And other porous materials. Of these, nitrocellulose membranes are preferred.

Here, the sample introducing section may be laminated on the support having the labeling section and the judging section. By doing so, a sufficient amount of the sample liquid can be supplied to the support on which the labeling section and the determination section are configured. Here, as the material of the sample introducing portion, for example, a water-absorbing porous material such as a non-woven fabric can be used.

Further, a water absorbing part for absorbing the sample liquid is laminated on the support, on which the labeling part and the determination part are formed, on the side opposite to the labeling part with respect to the determination part. Good. By doing this, it is possible to absorb the excess sample liquid on the support on which the labeling portion and the determination portion are configured. Here, examples of the material of the water absorbing portion include a porous material such as glass fiber filter paper.

The immune device of the present invention is preferably housed in a hollow case. Here, the hollow case may be made of, for example, plastic and at least the determination section and the sample introduction section are open, and the effect of preventing the sample liquid from leaking to the outside can be obtained.

Further, it is preferable that a material impermeable to the sample liquid is provided in close contact with at least a part of the sample introducing section, the labeling section and the judging section. For example, an adhesive tape made of a material impermeable to the sample solution may be attached to prevent the sample solution from leaking to the outside, and also to control the flow rate of the sample solution on the immune device and the flow of the sample solution. The effect of making uniform is obtained.

As the cross-linking substance for cross-linking two or more molecules of the test substance in the present invention, two reaction sites with the test substance are used.
One or more, for example, antibody, Fab, F
(Ab) ₂ , Fab ′, F (ab ′) ₂ , Fv, scF
v, compound containing antibody, compound containing Fab, F (a
b) a compound containing ₂ , a compound containing Fab ', F (a
b ′) ₂ containing compound, Fv containing compound, or sc
Examples thereof include compounds containing Fv. Among these, the crosslinking substance is preferably an antibody, F (ab) ₂ or a compound containing a plurality of Fabs.

The antibody used as a cross-linking substance for cross-linking two or more molecules of the test substance is preferably IgG or IgM because the binding valence is two or more. Alternatively, an antibody obtained by crosslinking two or more molecules of the antibody with a crosslinking reagent may be used. A compound containing a plurality of Fabs has two or more molecules of F (ab) ₂ or Fab.
Can be synthesized by crosslinking with a crosslinking reagent. Examples of the cross-linking reagent include DIDS, DPMDS, DTBPA, EGS, GM commercially available from Dojindo
BS, EMCS, HMCS, SPDP, etc. can be used.

Further, it is preferable that an optically detectable substance is bound to a cross-linking substance that cross-links two or more molecules of the test substance. By doing so, the detection signal indicating that the test substance is captured by the first specific binding substance can be enhanced. Examples of substances that can be optically detected include gold colloids, dye molecules, and fluorescent molecules. The size of the substance that can be optically detected is preferably small in order to eliminate the influence of steric hindrance, and in the case of particles, the particle size is preferably 50 nm or less.

It is preferable that the test substance has a plurality of reaction sites, and the reaction site to which the first specific binding substance and the second specific binding substance bind is different from the reaction site to which the cross-linking substance binds. By doing so, the effect of the present invention can be obtained even when the test substance is a monomeric protein.

Further, the test substance has three or more reaction sites, and the reaction sites with the first specific binding substance, the second specific binding substance and the cross-linking substance are not affected by steric hindrance. As described above, it is preferable that the positional relationships are apart from each other.
The test substance has three or more reaction sites, and the reaction sites are positioned so that the first specific binding substance, the second specific binding substance, and the cross-linking substance simultaneously bind to the test substance. More preferably. The positional relationship of the reaction sites can be confirmed by an antigen-antibody agglutination reaction or the like.
In addition, the antigenic determinant can be identified by using a biological substance interaction analysis technique based on the principle of surface plasmon resonance.

The cross-linking substance for cross-linking two or more molecules of the test substance in the present invention is preferably smaller than the complex of the sensitizing substance and the second specific binding substance. By doing so, as shown in FIG. 1, the second sensitized by the sensitizing substance is used.
Test substance 13 forming a complex with specific binding substance 15 of
Then, when the test substance 13 bound to the first specific binding substance 12 is crosslinked, the effect of being less susceptible to steric hindrance and facilitating the crosslinking reaction is obtained.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the drawings. The present invention is not limited to the following embodiments.

(Embodiment 1) h which is a test substance in urine
An immunochromatographic device for measuring CG will be described as an example.

(A) Selection of Antibody FIG. 2 is a schematic diagram showing the reaction binding site of the antibody which is the specific binding substance used in the present embodiment. hCG is
It has an α subunit 21 and a β subunit 22, and the first
The antibody used for immobilization, which is a specific binding substance of β, is β
Anti-hCG-β1 having reactive binding site in subunit 22
As the sensitizing antibody which is the second specific binding substance using the antibody 25, an anti-h having a reactive binding site in the α subunit 21 is used.
The CG-α1 antibody 23 is used. Further, the anti-hCG-β2 antibody 26 and the anti-hCG-α2 antibody 24 are used to prepare a cross-linking substance that cross-links two or more molecules of the test substance.

In order to confirm that the reaction sites in hCG differ among the above four types of antibodies, for example, S
The PR method can be used. Immobilizing hCG on a chemically modifiable gold thin film, anti-hCG-α1 antibody 23, anti-hCG-
α2 antibody 24, anti-hCG-β1 antibody 25, and anti-hCG
-Β2 antibody 26 is sequentially dropped on the hCG-immobilized gold thin film. FIG. 3 is a diagram showing an example of the result of measurement by the SPR method. In FIG. 3, the horizontal axis represents time and the vertical axis represents the vanishing angle of reflected light (the amount of antigen-antibody binding). As shown in the figure,
If the disappearance angle of reflected light (the amount of antigen-antibody binding) increases with the dropping of each antibody, it can be confirmed that each antibody has different reaction sites in hCG.

(B) Fab-making of anti-hCG-β2 antibody Fab-making of anti-hCG-β2 antibody can be carried out, for example, by the following steps.

5 mg / ml anti-hCG-β2 antibody solution 50
0 μl is dialyzed against acetate buffer (pH 4.5), 0.06 mg of pepsin is added, and the mixture is incubated at 37 ° C. for 24 hours. Adjust the pH to 7.0 and use Ultrogel ACA4
Gel filtration is performed using 4 columns (Pharmacia, 1.5 × 45 cm) (flow rate: 0.35 ml, eluent: 0.
1M phosphate buffer). F (ab ') ₂ thus obtained
0.1M mercaptoethylamine (phosphate buffer, p
H6.0) solution, 5 mM EDTA is further added, and the mixture is incubated at 37 ° C. for 90 minutes. Then S
Simple gel filtration was performed using ephadex G-25 (Pharmacia) (flow rate: 0.35 ml, eluent: 5 nM E
Fab '(DTA-0.1M phosphate buffer). After concentrating, the absorption characteristics (ε280 = 1.48)
L / g · cm) 1 mg / ml Fab ′ solution (phosphate buffer solution) is obtained.

(C) Preparation of cross-linking substance for cross-linking two or more molecules of test substance Preparation of cross-linking substance for cross-linking two or more molecules of test substance
For example, the following steps can be performed.

The SPDP represented by the following equation (Formula 1) is
Anti-hCG-β2 antibody Fab ′ and anti-hC prepared in (b)
Add 3 mg to the mixed solution of G-α2 antibody.

[0045]

[Chemical 1]

Anti-hCG-β2 antibody Fab ′ and anti-hCG-
Cross-linking reaction with α2 antibody is performed at 25 ° C for 30 minutes, then Se
Cross-linking substance (hereinafter referred to as “cross-linking substance” for cross-linking two or more molecules of the test substance shown in FIG. 4 is subjected to simple gel filtration with Phadex G-25 (Pharmacia) (flow rate: 0.35 ml, eluent 0.1 M phosphate buffer). , Anti-hCG-α2 antibody-anti-hCG-
β2 antibody Fab ′ cross-linking reagent) 1 mg / ml
Solution (phosphate buffer).

(D) Preparation of immunochromatographic device Next, preparation of an immunochromatographic device is performed. FIG. 5 shows the structure of the immunochromatographic device according to the present embodiment. Anti-hCG-β1 whose concentration was adjusted by diluting it with a phosphate buffer solution
Prepare antibody solution. This antibody solution is applied onto the nitrocellulose film 53, which is a support, using a solution discharge device. As a result, the antibody immobilization line that is the determination unit 54 is obtained on the nitrocellulose membrane 53. After drying this nitrocellulose membrane 53, it is immersed in a Tris-HCl buffer solution containing 1% skim milk and shaken gently for 30 minutes. After 30 minutes, the membrane was transferred to a Tris-HCl buffer solution tank and shaken gently for 10 minutes, and then another Tris-HCl buffer was added.
The membrane is washed by gently shaking for another 10 minutes in the HCl buffer solution tank. After washing twice, the membrane is removed from the liquid bath and dried at room temperature.

Gold colloid, which is a sensitizing substance, is 0.01%.
Chloroauric acid is prepared by adding a 1% citric acid solution to a 100 ° C. solution at reflux. Reflux is continued for 30 minutes and then left to cool at room temperature. To the gold colloid solution adjusted to pH 9 with a 0.2 M potassium carbonate solution,
After adding anti-hCG-α1 antibody and stirring for several minutes,
9% 10% BSA (Bovine Serum Albumin) solution to a final 1%
Then, an antibody-gold colloidal complex (sensitizing antibody), which is the second substance sensitized with the sensitizing substance, is prepared by adding and stirring the same amount. The sensitizing antibody solution was kept at 4 ° C for 200
The sensitized antibody was isolated by centrifugation at 00 G for 50 minutes, suspended in a washing buffer (1% BSA / phosphate buffer), and then subjected to the centrifugation to perform sensitization. The antibody is washed and isolated. The sensitizing antibody is suspended in the washing buffer and filtered through a 0.8 μm filter. here,
A reagent is prepared by adding an anti-hCG-α2 antibody-anti-hCG-β2 antibody Fab ′ crosslinking reagent to the sensitizing antibody so that the final concentration is 1 mg / ml.

The above-mentioned reagent is set in the solution discharge device and the nitrocellulose membrane 53 on which the anti-hCG-β1 antibody is immobilized.
After the coating is applied to a position away from the upper determination part 54, the film is dried to form the labeling part 51 on the nitrocellulose film 53. Further, in the nitrocellulose film 53, a portion on the opposite side of the determination portion 54 with the labeling portion 51 functioning as the sample introduction portion 52. Finally, by cutting the nitrocellulose membrane 53 into strips of 5 mm, the immunochromatographic device of this embodiment can be manufactured.

In the immunochromatographic device of the present embodiment thus produced, the determination unit 54 is determined by the hCG that does not form a complex with the sensitizing antibody in the antigen excess region.
Even when the anti-hCG-β1 antibody immobilized on Escherichia coli was blocked, hCG complexed with the sensitizing antibody and anti-hC
Since the hCG bound to the G-β1 antibody is crosslinked via the anti-hCG-α2 antibody-anti-hCG-β2 antibody Fab ′, which is a crosslinker, the sensitizer is immobilized on the determination unit 54. It is possible to suppress a decrease in signal strength.

[0051]

EFFECTS OF THE INVENTION According to the present invention, the detection of a test substance in a sample solution, which has a wide measurement range and few malfunctions, is suppressed by suppressing a decrease in signal intensity in an excessive amount range of the test substance. An immunoassay method, a kit and a device used therefor can be provided.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a cross-linking substance for cross-linking two or more molecules of a test substance according to the present invention and a reaction form thereof.

FIG. 2 is a schematic diagram showing a reactive binding site of an antibody used in one embodiment of the present invention.

FIG. 3 is a graph showing an example of the result of measurement by the SPR method for the antibody used in the embodiment of the present invention.

FIG. 4 is a structural diagram of a cross-linking substance used in one embodiment of the present invention to cross-link two or more molecules of a test substance.

FIG. 5 is a structural diagram of an immunochromatographic device according to an embodiment of the present invention.

FIG. 6 is a schematic diagram showing a sandwich type immune reaction in a conventional pregnancy diagnostic drug.

FIG. 7 is a graph showing the relationship between the amount of test substance and detection sensitivity in conventional pregnancy diagnostic agents.

FIG. 8 is a schematic diagram showing reaction states in three regions in which the amount of a test substance is different in a conventional pregnancy diagnostic drug.

[Explanation of symbols]

11,71 support 12 First specific binding substance 13 Test substance 14 Cross-linking substances 15 Second specific binding substance 21 hCG α subunit 22 β subunit of hCG 23 Anti-hCG-α1 antibody 24 Anti-hCG-α2 antibody 25 anti-hCG-β1 antibody 26 anti-hCG-β2 antibody 51 sign section 52 Sample introduction section 53 Nitrocellulose membrane 54 Judgment unit 72 Immobilized antibody 73 hCG 74 Sensitizing antibody 75 colored particles 81 First Area 82 Second Area 83 Third Area

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shigeto Osamu             1006 Kadoma, Kadoma-shi, Osaka Matsushita Electric             Sangyo Co., Ltd.

Claims (8)

[Claims] 1. A method for detecting or measuring a test substance in a sample solution, which comprises a step A of crosslinking two or more molecules of the test substance, which is immobilized on a support and specifically binds to the test substance. A step B of reacting a first specific binding substance that binds with the second specific binding substance that is sensitized with a sensitizing substance and that specifically binds with the test substance, and the sensitization By detecting or measuring the substance, the step B
The immunoassay method, which comprises the step C of detecting or measuring the complex containing the first specific binding substance, the second specific binding substance, and the test substance generated in (3). 2. A kit for detecting or measuring a test substance in a sample solution, comprising a support and a sensitizing substance to which a first specific binding substance that specifically binds to the test substance is immobilized. Second, which is sensitized by and binds specifically to the test substance
2. An immunoassay kit comprising a specific binding substance of 1. and a cross-linking substance that cross-links two or more molecules of the test substance. 3. A device for detecting or measuring a test substance in a sample liquid, comprising a sample introducing part, a labeling part and a judging part, wherein the labeling part is sensitized with a sensitizing substance, A second specific binding substance that specifically binds to the test substance is carried in a state that can be eluted by the sample solution, and the determination unit has a first specific binding that specifically binds to the test substance. A substance is fixed, the sample introducing unit, the labeling unit and the determination unit are arranged so that the sample solution introduced into the sample introduction unit moves to the determination unit via the labeling unit, and the sample An immune device comprising a cross-linking substance that cross-links two or more molecules of the test substance in a route until a liquid reaches the determination unit. 4. The cross-linking substance is an antibody, Fab, F (ab)
₂ , Fab ′, F (ab ′) ₂ , Fv, scFv, compound containing antibody, compound containing Fab, compound containing F (ab) ₂ , compound containing Fab ′, compound containing F (ab ′) ₂ The compound according to claim 3, which is a compound containing Fv, Fv, or a compound containing scFv. 5. The immune device according to claim 3, wherein the cross-linking substance includes a substance that can be optically detected. 6. The test substance has a plurality of reaction sites, the first
6. The immune device according to claim 3, wherein the reaction site to which the specific binding substance and the second specific binding substance are bound is different from the reaction site to which the cross-linking substance is bound. 7. The test substance has three or more reaction sites, and the first specific binding substance, the second specific binding substance, and the cross-linking substance simultaneously bind to the test substance, The said reaction site is located, The said 3 characterized by the above-mentioned.
The immune device according to any one of 6 above. 8. The immune device according to claim 3, wherein the cross-linking substance is smaller than the complex of the sensitizing substance and the second specific binding substance.