JPH04301765A - Immunological measuring method - Google Patents

Abstract

PURPOSE:To measure a specific component in a liquid sample accurately in a simple operation with a good reproducibility by soaking a good absorbent material in an immunization solution in a container, and measuring by transferring a mark body to an analytical element conjugated to the absorvent material. CONSTITUTION:By soaking an water absorvent material to which an analytical element 2 is conjugated beforehand in a reaction solution 4 in a container 3 after an immunization, a separate enzyme mark antibody (or an antigen) is transferred to the material 1 side by a capillary phenomenon. And by picking up the element 2 after a specific time has passed when the mark antibody has been transferred to the element 2 side further, the material 1 is removed from the element 2, and only the element 2 is picked up, and the mark body is measured. As the element 1, any substance can be used, while the element 2 is preferable to furnish porous layers more than one layer. And as a favarable example of the porous layer material, a structure composed of materials more than one selected from a granual body of the size about 1 to 350mum or a fiber of about 40 to 400 mesh is available.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method.

0002

BACKGROUND OF THE INVENTION Various analytical methods have been developed for detecting trace amounts of substances contained in biological fluid samples. One of these analytical methods is based on immune reactions. Various measurement methods using this principle have been developed and are known to be highly accurate. That is, since Berson and Yallow succeeded in measuring insulin in serum using bovine insulin labeled with radioisotope I and anti-insulin antibodies in the serum of diabetic patients, immunoassay methods using radioisotopes have been developed. It has been widely used. Since then, various labeling substances other than radioactive isotopes have been developed. Examples include enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, cycling reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, fluorescent molecules, and the like.

[0003] Such immunoassay methods are broadly classified into homogeneous immunoassay methods and non-uniform immunoassay methods. That is, the antigen-antibody reaction product (Bound body) and the non-reactant (Free
They are broadly divided into non-homogeneous immunoassay methods that require separation from the body (B/F separation) and homogeneous immunoassay methods that do not require B/F separation. Among these, when the substance to be measured is a polymer, a non-uniform immunoassay method that requires B/F separation is used.

[0004] However, this non-uniform immunoassay method has problems such as complicated operations because it requires washing operations, preparation of reagents, and addition of labeling substances, substrates, reaction stop solutions, etc. There is. Various techniques have been proposed to address these problems. For example, JP-A-64-6386
Publication No. 3, JP-A-64-63864, JP-A-2-
Although the operability of the method disclosed in Japanese Patent No. 8344 is considerably simplified by using a dry analytical element, the demand for simplicity continues to increase, and improvements are awaited.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a technique that allows accurate quantification of specific components in a liquid sample with good reproducibility using simple operations. The purpose of the present invention is to perform an immune reaction in a container, then immerse a water-absorbing material capable of exhibiting capillary action in the reaction solution in the container, and transfer the label to the water-absorbing material through this water-absorbing material. This is achieved by an immunoassay method characterized by analyzing a specific component in a sample by transferring the labeled substance to a bonded analytical element and measuring the labeled substance with the analytical element.

[0006] Also, after an immune reaction is performed in a container with the immobilized antibody (or antigen) immobilized on the immobilization carrier, the enzyme-labeled antibody (or antigen), and the antigen (or antibody) in the sample, analysis is performed. The water-absorbent material bonded to the element is immersed in the reaction solution, and the free enzyme-labeled antibody (or antigen) is transferred to the analytical element by capillary action of the water-absorbent material.Then, the analytical element is removed, and the labeled enzyme is transferred to the analytical element. This is achieved by an immunoassay method characterized by analyzing antigens (or antibodies) in a sample by measuring them.

The present invention will be explained in more detail below. In the present invention, any form of solution, colloidal solution, etc. can be used as the sample, but samples of biological origin are preferably used, such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid,
Examples include milk, urine, sweat, meat juice, etc. Examples of labeling substances used in immune reactions include enzymes, enzyme substrates, substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, apoenzymes, and fluorescent substances. Examples include β-D-galactosidase, alkaline phosphodase, peroxidase,
Enzymes such as glucose oxidase, glutamate dehydrogenase, and amylase are preferred, and when these enzymes are used as labeling substances, known enzyme reaction systems and coloring systems can be used. Specifically, JP-A-61-292060, JP-A-62-90539, JP-A-63-1
Examples include substances (substance groups) described in Japanese Patent Application Laid-Open No. 31062, Japanese Patent Application Laid-Open No. 63-45562, and Japanese Patent Application No. 63-219893, but are not limited thereto. The binding of the labeled substance to the antibody (antigen) can be carried out using known reagents and methods known to those skilled in the art. 2nd edition), Igaku Shoin, 1978" and "Immunoassays for Clinical Tests - Techniques and Applications -, Clinical Pathology Publications Society, 1983", Special Special Issue No. 53 of "Clinical Pathology" edited by the Japanese Society of Clinical Pathology, etc. You can refer to the method provided.

[0008] The origin of the antibody used in the immune reaction of the present invention is not particularly limited. Antiserum obtained by administering and immunizing a mammal with an antigen, ascites fluid as it is, or conventional Known methods such as sodium sulfate precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc. (Shunsuke Migita, "Immunochemistry", Nakayama Shoten p.
(see pages 74 to 88). Alternatively, hybrid cells (hybridoma) are obtained from spleen cells or myeloma cells (myeloma) of a mammal (e.g. mouse) infected with an antigen, and a monoclonal antibody is created, which is then used as a substance that can specifically bind to a specific component. It is preferable to use it as the specificity improves. Furthermore, these antibodies can be used in IgG, IgM, IgA, IgD, and IgE fractions, or these antibodies can be treated with enzymes to produce F.
It may also be used in the form of active antibody fragments such as ab, Fab' or F(ab')2. Furthermore, these antibodies may be used alone or in combination.

[0009] Reaction types according to the immunoassay method of the present invention include a competitive method, a two-antibody method, a sandwich method, etc., but are not particularly limited. Also, other biologically active substances (
For example, immunoassay methods using biotin, avidin) can also be applied. In the present invention, an immune reaction is mentioned as a reaction type for measuring a specific component in a sample, but a specific reaction of a substance that exhibits biological activity similar to an immune reaction (in this specification, this specific reaction is also included in the immune reaction). ) can also be used. Examples of combinations of substances that specifically bind include the following.

Enzyme and Substrate (Product) Enzyme and Inhibitor Enzyme and Prosthetic Group Enzyme and Coenzyme Enzyme and Allosteric Effector Antibody and Antigen Antibody and Protein A Lectin and Polysaccharide Lectin and Glycoprotein Nucleic Acid and Complementary Base Sequence Nucleic Acid and Histone Nucleic Acids and Nucleic Acids Nucleic Acids and Polymerases Hormones and Receptors Pyotin and Avidin (Streptavidin) Bioticin and Avidin (Streptavidin) Desthiobiotin and Avidin (Streptavidin) Oxybiotin and Avidin (Streptavidin) Antigens Used in the Invention reacts with specific antibodies and contains haptens and derivatives thereof.

[0011] The antibody (or antigen) is immobilized on an insolubilizing carrier. The insolubilizing carrier is a granular material, for example, a size of 2 m.
Particles with a diameter of m or less are preferable. Materials for the insolubilizing carrier (fine particles) include agarose, cellulose, cross-linked dextran, polyacrylamide, cellulose, microcrystalline cellulose, cross-linked agarose, cross-linked polyacrylamide, glass, silica gel, diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, In addition to various synthetic resins such as lead oxide, silica sand, and polystyrene, porous materials and even magnetic particles can be used. Preferably agarose, cross-linked agarose,
Examples include crosslinked dextran, polyacrylamide, crosslinked polyacrylamide, glass, silica gel, polystyrene, cellulose, microcrystalline cellulose, and more preferably polyacrylamide, crosslinked polyacrylamide, polystyrene, microcrystalline cellulose, and the like. Several types of these insolubilizing carriers may be used in combination. Antibodies or antigens can be chemically and/or physically bound to these insolubilized carriers directly or indirectly by methods known to those skilled in the art. Regarding binding methods, ``Experiments and Applications Affinity Chromatography'' (1st edition) published by Kodansha, 1975, edited by Makoto Yamazaki and 2 authors, published by Kodansha, ``Experiments and Applications Affinity Chromatography'' (1st edition) version) can be used as a reference. After the binding reaction, labeled antibody (
In order to eliminate non-specific reactions (or antigens), proteins that are not involved in the specific reaction to be measured can be supported. Typical examples thereof include normal serum proteins of mammals and birds, albumin, skim milk, lactic acid fermentation products, collagen, and their decomposed substances. In addition, the non-specific adsorption-inhibiting protein is not only supported on the insolubilized carrier, but also added in a certain amount to the immune reaction solution during the immune reaction, thereby further increasing the effect of suppressing non-specific adsorption.

[0012] Then, the above-mentioned immobilized antibody (or antigen), enzyme-labeled antibody (or antigen), and antigen (or antigen) in the sample are
After the immunoreaction with enzyme-labeled antibodies (or antigen) will migrate to the water-absorbing material side. Note that any water-absorbing material may be used.

[0013] An analytical element is bonded to this water-absorbing material. As shown in FIG. 1, the water-absorbing material 1 and the analytical element 2 that are integrated in advance are dropped into the container 3 after the immune reaction, and the water-absorbing material 1 is immersed in the reaction solution 4, and the water-absorbing material 1 is immersed in the reaction solution 4, and the water-absorbing material 1 is immersed in the reaction solution 4. When the analytical element 2 is pulled up a predetermined time after the free enzyme-labeled antibody (or antigen) has migrated to the water-absorbent material 1 side and further migrated to the analytical element 2 side, the water-absorbent material 1 is peeled off from the analytical element 2 and analyzed. In some cases, only the element 2 can be lifted up, or as shown in FIG. Due to the capillary action of the absorbent material 1, free enzyme-labeled antibodies (or antigens) are transferred to the water absorbent material 1 side.
Furthermore, after a predetermined period of time after moving to the analytical element 2 side, the analytical element 2
It is fine as long as it is able to raise the

The analytical element used in the present invention preferably has at least one porous layer. The material of the porous layer is not particularly limited, but a preferred example is size 1.
Examples include structures made of one or more materials selected from granules of 350 μm to 350 μm or fibers of 40 to 400 mesh. Materials for the granules include diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, cross-linked dextrin, cross-linked polyacrylamide, agarose, cross-linked agarose, polystyrene, etc. In addition to various synthetic resins, poly(styrene-glycidyl methacrylate),
Examples include self-bonded particles made of compounds with reactive groups such as poly(styrene-methyl acrylate-glycidyl methacrylate).

[0015]Fibers used for the porous layer include pulp, powder filter paper, cotton, hemp, silk, wool, chitin, chitosan,
Cellulose ester, viscose rayon, copper ammonia rayon, polyamide (6-nylon, 6,6-nylon, 6,10-nylon, etc.), polyester (polyethylene terephthalate, etc.), polyolefin (polypropylene, vinylon, etc.), glass fiber, asbestos Vegetable, animal, synthetic, semi-synthetic, and regenerated fibers such as fibers may be used, or a mixture of these may be used. Alternatively, water-absorbing Western paper, Japanese paper, filter paper, brushed polymer, glass fiber, mineral fiber (asbestos, etc.), vegetable fiber (cotton, hemp, pulp, etc.), animal fiber (
wool, silk, etc.), synthetic fibers (various nylons, vinylon,
Woven fabrics, nonwoven fabrics, synthetic papers, etc. made of polyethylene terephthalate, polypropylene, etc.), recycled fibers (rayon, cellulose ester, etc.) singly or in combination can also be used for the porous layer.

[0016] By applying and/or forming a film from such granules, fibers, or a mixture of granules and fibers,
A porous layer is formed in which there is a porous structure with freely accessible interconnecting pores. Particles that do not have self-bonding properties can be formed into a film by point-adhering the particles to each other using a suitable adhesive. Kaisho 57-67
The method described in Japanese Patent No. 860 can be applied. Organic polymer particles with self-bonding properties are disclosed in Japanese Patent Application Laid-open No. 57-10.
Films can be similarly formed by the methods described in JP-A No. 1760, JP-A-57-101761, and JP-A-58-70163. For fibers or mixtures of fibers and particles, see JP-A-57-125847 and JP-A-57-197.
A porous layer can be formed by applying the fiber dispersion described in Japanese Patent No. 466. Also, JP-A-60-1734
It is also possible to apply a fiber dispersion using a water-soluble binder such as gelatin or polyvinylpyrrolidone, as in the method used in Japanese Patent No. 71. Further, the binder at this time is not limited to a water-soluble one, and a hydrophobic binder can also be used. Many methods can be used alone or in combination to produce such dispersions. For example, one useful method is to add surfactants to the liquid carrier to promote distribution and stabilization within the dispersion of particulates and/or fibers.

Examples of typical surfactants that can be used include Triton X-100 (manufactured by Rohm and Haas Co., Ltd.).
There are nonionic surfactants such as octylphenoxypolyethoxyethanol) and Surfactant 10G (manufactured by Orin Co., Ltd., nonylphenoxypolyglycidol). These surfactants can be used in widely selected amounts, but at least 0% based on the weight of the granules and/or fibers.
．． 0.005 to 10% by weight, preferably 0.15 to 6% by weight. Furthermore, as another method,
sonication, physical mixing of the particle units and the liquid carrier;
and physical stirring treatment and pH adjustment. These are even more useful in combination with the methods described above.

The form of the dry analytical element is not particularly limited as long as it can perform analysis, but from the viewpoint of manufacturing and measurement operations, it is preferably in the form of a film or a sheet. Even though a dry analytical element consists of a single layer,
It may also consist of multiple layers. For example, one may consist of only a porous layer containing an enzyme substrate, one may have a porous layer on top of a water absorption layer (the substrate is contained in at least one of the layers), or one may have multiple porous layers on top of a water absorption layer. The substrate may be provided with layers (the substrate is incorporated in at least one of the layers), and if necessary, they may be provided on a light-transmitting support or on a light-reflecting support. Alternatively, it may be provided on a light-transmitting support and a light-reflecting layer may be provided thereon.

Examples of materials for the water absorption layer include gelatin, gelatin derivatives such as phthalated gelatin, synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, and sodium polyacrylate, hydroxyethyl cellulose, and carboxymethyl cellulose. Examples include polysaccharides of cellulose derivatives such as sodium salts. Preferred are gelatin derivatives such as gelatin and phthalated gelatin.

Materials for the light-transmitting support include, for example, cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds (such as polystyrene).
Examples include transparent polymer compounds such as , and transparent inorganic compounds such as glass. Materials for the light-reflective support include, for example, ceramics, metals, or resin-coated paper, and if necessary, these materials may contain or be coated with white pigments such as TiO2, BaSO4, or mica. It's okay to let them do it.

[0021] For example, when a dry analytical element is used, in which a porous layer containing a substrate is provided on a light-transmitting support, a dry analytical element is applied to the water-absorbing material immersed in the immunoreaction solution. The porous layer side of the analytical element is bonded, and the free enzyme-labeled antibody (or antigen) migrates to the porous layer of the dry analytical element through the water-absorbing material, so that color signals are measured from both sides. . In addition, when a dry analytical element is used, in which a porous layer containing a substrate is provided on a light-reflective support, the pores of the dry analytical element are Since the dry layer side is joined and the free enzyme-labeled antibody (or antigen) is transferred to the porous layer of the dry analytical element via the water-absorbing material, signal measurement is performed from the porous layer side. In addition, when a dry analytical element is used in which a porous layer containing a substrate is provided on a light-transmitting support and a light-reflecting layer is provided on top of the porous layer, the water-absorbing material is the light-reflecting layer. The sides are bonded and the signal is measured from the side of the light-transparent support.

[0022] Signals caused by labels include ultraviolet light, visible light,
It can be detected by absorbance method (colorimetric method) using near-infrared light, etc., and measurement methods include rate measurement method that measures changes in the signal over time or end point measurement that measures the signal after a certain period of time. It can be measured by the method. The dry analytical element may have a spreading layer. As the material for the spreading layer, the same material as that for the porous layer may be applied, formed into a film, or attached. In addition, by incorporating a substrate etc. into the developing layer,
It may also serve as a reaction layer. Dry analytical elements may contain other additives such as buffers, preservatives, surfactants,
A mordant or the like can be added depending on the purpose. The buffer is a pH suitable for specific binding reactions, enzymatic reactions, color reactions, etc.
Contained to make H. Buffers that can be used include "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd. 1966) pp1312-1320, N
．． E. Good et al; Biochemistry V
ol 5, p467 (1966), Imamura, Saito; Chemistry Area, Vol 30 (2), p79 (1976), W
．． J. Ferguson et al. Anal. Bioche
m. Examples include those described in literature such as Vol 104, p300 (1980). Specific examples include borates, citrates, phosphates, carbonates,
Examples include trisbarbiturate, glycine, and Good's buffer. These buffering agents may be used alone to form a layer, if necessary. Preservatives are included to stabilize the storage of the substrate coloring reagent, and include antioxidants and the like. The substance is described in "Biochemistry Experiment Account 1, Protein Chemistry 1" edited by the Japanese Biochemical Society (Tokyo Kagaku Doujin Co., Ltd. 1976) pp66
, 67, Experiments and Applications "Affinity Chromatography"
Examples include those described in pp. 16 to 104, JP-A-60-149927, and the like. Specific examples include gelatin, gelatin decomposition products, albumin, cyclodextrins, non-reducing sugars (sucrose, trehalose), polyethylene glycol, amino acids, various ions, sodium azide, and the like. Examples of the surfactant include those mentioned above. Other reagents contained in the layer include solubilizers, blocker reagents, and the like. These additives are added in appropriate amounts as necessary. A mordant is a substance that concentrates the detection substance for enzyme activity measurement on the photometer side, increases the absorbance coefficient when the detection substance is a dye, or shifts the wavelength, and exhibits strong interaction with the detection substance. . Cationic polymers, anionic polymers and latexes of these polymers are used. The dry analytical element can further be provided with auxiliary layers such as a blood cell separation layer useful when the biological sample is blood (whole blood), an adhesive layer, a protective layer, and a timing layer provided as necessary. The positions of these layers are determined according to their functions.

[0023]

EXAMPLES The present invention will be explained in more detail below with reference to Examples, but the present invention is not limited to these Examples. 1-(1) Preparation of β-D-galactosidase-labeled CRP antibody 20 mg of CRP antibody (rabbit IgG fraction, manufactured by Townes) was dissolved in 0.1 M phosphate buffer (p6.5)2.
0 ml of N-(ε-maleimidocaproyloxy)succinimide (manufactured by Dojindo Laboratories).
After adding 77 μl of 5 mg/ml dimethylformamide solution and reacting at 30°C for 20 minutes, it was applied to a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA. A purified and maleimidated CRP antibody was obtained.

Next, β-D-galactosidase (manufactured by Toyobo) was added to 0.1M phosphate buffer solution 1 containing 10.5 mg/ml.
．． 13. Add the maleimidated CRP antibody to 8 ml.
Add 3.2 ml of a solution containing 6 mg and react at 4°C for 45 hours, then add 175 μ of 0.1 M 2-mercaptoethylamine.
1 was added and reacted at 30°C for 20 minutes, and then diluted with 0.1M phosphate buffer (pH 7
．． 4) was separated and purified using a Superrose 6 prep grade (manufactured by Pharmacia) column, and β-D-
A galactosidase-labeled CRP antibody was obtained.

1-(2) Synthesis of CRP antibody-immobilized Eupergit C 3 g of Eupergit C (manufactured by Rohm Pharma) was mixed with 0.1
It was dispersed in 40 ml of 0.1 M phosphate buffer (pH 8.0) containing 5 M sodium chloride, and 136 ml of CRP antibody (rabbit IgG fraction, manufactured by Townes) was added to it.
g and stirred at 4°C for 20 hours to react. After the reaction,
Collected by filtration, mixed with 0.1M acetate buffer (pH 4.0) and 0.1M
The carbonate buffer solution (pH 8.0) was used alternately to wash thoroughly.

[0026] After washing with water, the mixture was sieved through a 38 μm mesh. In order to block non-specific binding sites of Eupergit C, the sieved Eupergit C was stirred in 50 ml of 0.1 M Bis Tris buffer (pH 7.2) supplemented with 3% skim milk at 4°C for 20 hours. . Then, it was washed with water, and CRP antibody-immobilized Eupergit C was added.
I got it.

1-(3) Creation thickness of dry analytical element 1
Coating solution (1) with the following composition was applied onto a transparent subbed polyethylene terephthalate film of 80 μm,
It was dried to form a gelatin layer. Coating liquid - (1) Deionized gelatin
6.0g Triton X-100
0.15g 1,2-bis(vinylsulfonyl)ethane 0.01g Pure water
54.0 g Next, coating liquid (2) was applied onto the gelatin layer and dried.

Coating liquid-(2) Powder filter paper C (manufactured by Toyo Roshi Co., Ltd.)
21.6g Pyrrolidone-vinyl acetate (2:8) copolymer 11.0g Chlorophenyl red β-D-galactopyranoside (manufactured by Boehringer)

365mg n-butanol
49.4
g This was cut into a size of 1.5 cm x 1.5 cm to form a dry analysis element, and a cylindrical pulp body (diameter 5 mm, height 10 mm) was glued onto this dry analysis element as shown in Figure 1. .

1-(4) Measurement of CRP 1mM Mg
Anti-CRP immobilized Eupergit C15 in 190 ml of 0.5 M Bis Tris buffer containing Cl2 and 3% BSA.
mg, β-D-galactosidase labeled CRP antibody (20
25 μl of μg/ml) and CRP solution (0.3, 10,
Add 7 μl of 30, 100, 300 μg/ml), mix, and allow immunoreaction for 12 minutes at room temperature. After the reaction, the cylindrical pulp of the dry analytical element was immersed in the reaction solution, and after 5 seconds, the dry analytical element was lifted and the reflection density at 546 nm was measured while incubating it at 37°C. 3 minutes 30
FIG. 3 shows the results of creating a calibration curve using the reflection density difference (ΔDr) from seconds to seven minutes.

[0030] According to this method, it has been found that CRP can be measured accurately, and the measurement can be carried out simply by dropping the water-absorbing material and the analytical element into the reaction solution. That is, the background noise is small, CRP can be measured accurately, and the measurement can be performed without any special operation for B/F separation or liquid transfer operation.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the immunoassay method of the present invention.

FIG. 2 is a schematic diagram showing the immunoassay method of the present invention.

FIG. 3 is a graph showing a calibration curve.

Claims (2)

[Claims] Claim 1: After an immune reaction is carried out in a container, a water-absorbing material capable of exhibiting capillary action is immersed in the reaction solution in the container, and the label is bonded to the water-absorbing material via this water-absorbing material. An immunoassay method characterized in that a specific component in a sample is analyzed by transferring the labeled substance to an analytical element and measuring the labeled substance with the analytical element. 2. After performing an immune reaction in a container with the immobilized antibody (or antigen) immobilized on the immobilization carrier, the enzyme-labeled antibody (or antigen), and the antigen (or antibody) in the sample, analysis is performed. The water-absorbent material bonded to the element is immersed in the reaction solution, and the free enzyme-labeled antibody (or antigen) is transferred to the analytical element by capillary action of the water-absorbent material.Then, the analytical element is removed, and the labeled enzyme is transferred to the analytical element. An immunoassay method characterized by analyzing antigens (or antibodies) in a sample by measuring.