JPH04351963A - Immunological measurement device and method thereof - Google Patents

Abstract

PURPOSE:To carry out quantitative analysis on a specific component in a sample accurately in a simple operation by providing a material capable of presenting capillarity so as to clog an opening in the lower part of a container, and inputting the sample in a place where an antibody is fixed onto the material. CONSTITUTION:In terms of operational simplicity, an enzyme labeling antibody is put in a container beforehand together with a stabilized/insoluble carrier. The antibody is stabilized in the insoluble carrier. The antibody carries out immunization reaction in the container 1 by means of the insoluble carrier 4, the enzyme labeling antibody and an antibody in a sample. After a prescribed time, an analytical element is brought into contact with a part of glass wool 2 exposed from an opening 3 in the bottom surface part of the container 1. The free state enzyme labeling antibody transmitted through layers of the carrier 4 and the glass wool 2 moves to the analytical element side, so that immunological analysis can be carried out. In order to move the free state enzyme labeling antibody swiftly to the analytical element side, the surface layer of analytical element is required to be given a water absorptive property. Accordingly, a porous layer is formed.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological measuring device and an immunological measuring 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.

[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 simple operations and good reproducibility. The object of the present invention is to provide a container;
An opening formed in the lower part of this container, a substance exhibiting capillary action arranged so as to close this opening, and an antibody (or antigen) arranged in the upper part of this material is immobilized. This is achieved by an immunoassay device characterized by comprising an insoluble carrier.

[0006] The present invention also includes a container, an opening formed in the lower part of the container, a substance exhibiting capillary action disposed so as to close the opening, and an antibody ( This is achieved by an immunoassay device characterized by comprising an insoluble carrier on which a labeled antibody (or antigen) is immobilized, and a labeled antibody (or antigen) placed in the container. The shape of the container in which the immune reaction of the present invention is carried out may be any shape, as long as an opening (for example, a hole) is formed in at least a portion of the container, for example, a portion of the bottom surface. . This opening is blocked with a substance that exhibits capillary action, such as cotton or glass wool, to prevent the sample from flowing out under normal conditions even if the container is filled with a fluid sample. It is composed of

[0007] As the sample to be measured by the immunoassay device of the present invention, any type of solution, colloid solution, etc. can be used, but samples of biological origin are preferably used, such as blood, plasma, serum, cerebrospinal fluid, saliva, etc. Examples include amniotic fluid, milk, urine, sweat, meat juice, etc. Enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants as labeling substances for antibodies (or antigens) used in the present invention and fluorescent molecules, such as β-D-galactosidase, alkaline phosphodase, peroxidase, glucose oxidase, glutamate dehydrogenase,
Enzymes such as amylase are preferred. 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-63-131,
Examples include, but are not limited to, substances (substance groups) described in Japanese Patent Application Publication No. 062, Japanese Patent Application Laid-Open No. 63-45562, and Japanese Patent Application No. 63-219893. The binding of these labeling substances to antibodies (antigens) can be carried out using known reagents and methods known to those skilled in the art.
For example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai (eds.)
"Enzyme immunoassay (2nd edition), Igaku Shoin, 1978" and "Immunoassay for clinical examinations - technology and applications", "Clinical Pathology" Special Special Issue No. 53, edited by the Japanese Society of Clinical Pathology, "Immunoassay for clinical tests - technology and applications", Clinical Pathology Publishing Society, 1983" can be referred to.

[0008] The enzyme-labeled antibody (or antigen) only needs to be present in the container during the immune reaction, and from this point of view it may be present when adding the liquid sample, but if the antibody (or antigen) is present in the container in advance
It is preferable that an insoluble carrier having immobilized thereon (or antigen) be included together with the insoluble carrier is preferable from the viewpoint of ease of measurement operation. The origin of the antibodies used in the present invention is not particularly limited. Antiserum obtained by administering and immunizing mammals with antigens, ascites fluid as is, or sulfuric acid using a conventionally known method. Sodium precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion exchange cellulose chromatography method, electrophoresis method, etc.
It can be purified and used by "Immunochemistry" Nakayama Shoten pp. 74-88). 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. In addition, these antibodies include IgG, IgM, IgA, IgD,
IgE fractions can be used or these antibodies can be enzymatically treated to produce Fab, Fab' or F(ab')2.
It may also be used in the form of active antibody fragments. Furthermore, these antibodies may be used alone or in combination.

[0009] Reaction types using the immunoassay device of the present invention include a competitive method, a two-antibody method, a sandwich method, etc., but are not particularly limited. Furthermore, immunoassay methods using other biologically active substances (eg, biotin, avidin) can also be applied. The antigen used in the present invention reacts with specific antibodies and includes haptens and derivatives thereof.

[0010] The antibody (or antigen) is immobilized on an insoluble carrier. The insoluble carrier is preferably granular, but is not limited thereto. Materials for the insoluble carrier include agarose, cellulose, cross-linked dextran, polyacrylamide, cellulose, microcrystalline cellulose, cross-linked agarose, cross-linked polyacrylamide, glass, silica gel,
In addition to various synthetic resins such as diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, silica sand, and polystyrene, porous materials and even magnetic fine particles can be used. Preferably agarose, crosslinked agarose, crosslinked dextran, polyacrylamide, crosslinked polyacrylamide,
Examples include glass, silica gel, polystyrene, cellulose, microcrystalline cellulose, and more preferably polyacrylamide, crosslinked polyacrylamide, polystyrene, microcrystalline cellulose, and the like. Several types of these insoluble carriers may be used in combination. Antibodies or antigens can be chemically and/or physically coupled to these insoluble carriers directly or indirectly by methods known to those skilled in the art. Regarding binding methods, ``Experiments and Applications Affinity Chromatography'' published by Kodansha in 1976, edited by Ichiro Chibata and two others.
(1st printing), 1975, published by Kodansha, edited by Makoto Yamazaki and 2 others, “Affinity Chromatography” (1st edition)
can be used as a reference. After the binding reaction, proteins that are not involved in the specific reaction to be measured can be supported in order to eliminate non-specific reactions of the labeled antibody (or antigen). 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 an insoluble carrier;
By adding a certain amount of it to the immune reaction solution during the immune reaction, the effect of suppressing non-specific adsorption is further enhanced. still,
This insoluble carrier is placed on top of the capillary substance in the container.

[0011] Then, the antibody (or antigen) as described above
Insoluble carrier immobilized with enzyme-labeled antibody (or antigen)
After the immune reaction by the antigen (or antibody) in the sample is carried out in the container described above, the cotton or glass wool part exposed from the opening at the bottom of the container is brought into contact with the analytical element after a predetermined period of time. For example, the free enzyme-labeled antibody (or antigen) that has permeated the insoluble carrier and glass wool layer will migrate to the analytical element side, and immunological analysis will be performed.

This analytical element contains, for example, an enzyme substrate, and in order to quickly transfer free enzyme-labeled antibodies (or antigens) to the analytical element, the surface layer needs to have water-absorbing properties. One example for this purpose is to form a porous layer. The material of the porous layer is not particularly limited, but a preferred example is a size of 1 to 350 μm.
Examples include structures made of one or more materials selected from granular materials 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 synthetic resins, examples include self-bonding particles made of compounds having reactive groups such as poly(styrene-glycidyl methacrylate) and poly(styrene-methyl acrylate-glycidyl methacrylate). Fibers include pulp, powdered filter paper, cotton, linen, 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.), Vegetable, animal, synthetic, semi-synthetic, and regenerated fibers such as glass fiber and asbestos 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 fibers (wool, silk, etc.), synthetic fibers (various types of nylon, vinylon, polyethylene terephthalate, polypropylene, etc.), recycled fibers (rayon, cellulose ester, etc.), etc. Woven fabrics, non-woven fabrics, and synthetic papers manufactured either singly or in combination etc. can also be used for the porous layer.

[0013] By applying and/or forming a film from such granules, fibers, or a mixture of granules and fibers,
A porous layer can be 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 57-6
The method described in Japanese Patent No. 7860 can be applied. Organic polymer particles with self-bonding properties are disclosed in Japanese Patent Application Laid-Open No. 57-1
No. 01760, JP-A-57-101761,
A film can be similarly formed by the method described in JP-A-58-70163. For fibers or mixtures of fibers and particles, see JP-A-57-125847 and JP-A-57-1.
A porous layer can be formed by applying the fiber dispersion described in Japanese Patent No. 96466. Also, JP-A-60-17
It is also possible to apply a fiber dispersion using a water-soluble binder such as gelatin or polyvinylpyrrolidone, as in the method practiced in Japanese Patent No. 3471. Moreover, 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 non-containing surfactants such as Triton There are ionic surfactants. These surfactants can be used in widely selected amounts, but less than 0.0% based on the weight of the granules and/or fibers
05 to 10% by weight, preferably 0.15 to 6% by weight. Furthermore, other methods include ultrasonication, physical mixing, and physical agitation of the particle units and the liquid carrier, and pH adjustment. These are even more useful in combination with the methods described above.

The shape of the 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. The analytical element may consist of a single layer or may 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 a water-absorbing layer (the substrate is contained in at least one of the layers), or one may have a porous layer on a water-absorbing layer (the substrate is contained in at least one of the layers), or a porous layer on a water-absorbing layer. Porous layers (with a substrate built into at least one of the layers) may be provided, 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-absorbing layer include gelatin, gelatin derivatives such as phthalated gelatin, synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylimidazole, polyacrylamide, and sodium polyacrylate, hydroxyethylcellulose, and carboxylic acid. Examples include polysaccharides of cellulose derivatives such as methylcellulose sodium salt. 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. Examples of materials for the light-reflective support include ceramics, metals, and resin-coated paper, and if necessary, these materials may contain or be coated with white pigments such as TiO2, BaSO4, or mica. It's fine if you let it happen.

For example, when an analytical element comprising a porous layer containing a substrate on a light-transmissive support is used, the color signal generated by the free enzyme-labeled antibody (or antigen) can be measured on both sides. It is possible to do it from Furthermore, when an analytical element is used in which a porous layer containing a substrate is provided on a light-reflective support, the signal is measured from the porous layer side. In addition, when using an analytical element 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 signal is measured on the light-transmitting support side. It is carried out from

Signals caused by labels include ultraviolet light, visible light,
It can be detected by absorbance method (colorimetric method) using near-infrared light, etc. Measurement methods include rate measurement method, which measures changes in the signal over time, or endpoint measurement, which measures the signal after a certain period of time. It can be measured by the method. Other additives such as buffers, preservatives, surfactants, mordants, etc. can be added to the analytical element depending on the purpose. A buffer is included to maintain a pH suitable for specific binding reactions, enzyme reactions, coloring reactions, and the like. Buffers that can be used are described in "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd. 1966) pp1312-1320, N
,E,Good;Biochemstry Vol.
5, p467 (1966), Imamura, Saito; Chemistry Area, Vol 30 (2), p79 (1976), W. J. F
Anal. Biochem. Vo
Examples include those described in literature such as 1104, p300 (1980). As a specific example,
Examples include borate, citrate, phosphate, carbonate, trisbarbiturate, glycine, Good's buffer, and the like. These buffering agents may be used alone to form a layer, if necessary. Preservatives are contained, for example, for storage stabilization of the substrate coloring reagent, and include antioxidants and the like. Examples of such substances include "Biochemistry Experiment Account 1, Protein Chemistry 1" (Tokyo Kagaku Doujin Co., Ltd. 1976), edited by the Japanese Biochemical Society, pp. 66 and 67, and "Affinity Chromatography", pp. 16, "Experiments and Applications".
-104, and those described in Japanese Patent Application Laid-Open No. 149927/1983. 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 contained reagents include solubilizing agents and blocker reagents. 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.

[0020]

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited by the Examples. [Example 1] 1-(1) Preparation of β-D-galactosidase-labeled anti-CRP antibody 20 mg of anti-CRP antibody (rabbit IgG fraction, manufactured by Townes) 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 maleimidized anti-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.
．． Add 13 ml of the maleimidated anti-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 0.1 M 2-mercaptoethylamine 175
μl of 0.1M phosphate buffer containing 0.15M sodium chloride (pH
The β-D
- Galactosidase-labeled anti-CRP antibody was obtained.

1-(2) Preparation of anti-CRP antibody-immobilized Eupergit C 3 g of Eupergit C (manufactured by Rohm Pharma) was mixed with 0.1
Anti-CRP antibody (rabbit IgG fraction, manufactured by Townes) 136 was dispersed in 40 ml of 0.1 M phosphate buffer (pH 8.0) containing 5 M sodium chloride.
mg and stirred at 4°C for 20 hours to react. After the reaction, it was collected by filtration and mixed with 0.1M acetate buffer (pH 4.0).
Thorough washing was performed by alternately using 1M carbonate buffer (pH 8.0). Next, after washing with water, Eupergit C was added at 3% to block the non-specific binding site of Eupergit C.
0.1M Bis Tris buffer (pH 7.2) with % skimmed milk
) 50 ml and stirred at 4°C for 20 hours. Next, it was washed with water and lyophilized to obtain anti-CRP antibody-immobilized Eupergit C.

1-(3) Preparation of immunological reaction container As shown in FIG. 1, the lower part of a plastic container 1 is filled with glass wool 2, the hole 3 is plugged, and the anti-CRP is placed on top of the glass wool 2. Antibody immobilized Eupergit C4 20
I put in mg. Next, 450 μl of the β-D-galactosidase-labeled anti-CRP antibody solution (10 μg/ml) was added and then freeze-dried to form an immunological reaction container.

1-(4) Preparation of analytical element (water-absorbing element) Coating liquid (1) having the following composition was applied onto a transparent subbed polyethylene terephthalate film with a thickness of 180 μm, dried, and gelatin Created a 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 prepare an analytical element.

1-(5) Measurement of CRP 30 μl of 0.3M Bis Tris buffer containing 1mM magnesium chloride and 3% by weight bovine serum albumin and CRP
Solution (0.1, 3, 10, 30, 100 μg/ml) 5
μl was placed in the immunological reaction container and subjected to immunoreaction for 10 minutes.

After this, the immunological reaction container is brought close to the analytical element, the glass wool 2 is brought into contact with the analytical element, and then the immunological reaction container is moved away from the analytical element. and,
After incubation at 37°C, the reflection density at 546 nm was measured from the support side of the analytical element. A calibration curve was created using the reflection density difference (ΔDr) for 3 minutes 30 seconds and 7 minutes, so
The results are shown in FIG. Note that FIG. 2 shows a CRP calibration curve created by the apparatus of the present invention.

[0028] According to this method, it has been found that CRP can be measured accurately, and the measurement can be performed with extremely simple and easy work. That is, background noise is small and CRP
can be measured accurately, and the measurement can be performed without complicated operations for B/F separation or liquid transfer operations.

[0029]

[Effect] According to the present invention, with simple and convenient operation,
That is, the measurement can be carried out with simple operations without complicated operations for B/F separation or liquid transfer operations, and the measurement results have low background noise and are accurate.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the principle of an immunological measuring device of the present invention.

FIG. 2 shows a calibration curve of CRP prepared according to the present invention.

[Explanation of symbols]

1. Plastic container 2 Glass wool 3 holes

Claims (3)

[Claims] Claim 1: A container, an opening formed in the lower part of the container, a substance exhibiting capillary action disposed so as to close the opening, and an antibody ( or an insoluble carrier on which an antigen) is immobilized. 2. A container, an opening formed in the lower part of the container, a substance exhibiting capillary action disposed so as to close the opening, and an antibody ( An immunoassay comprising: an insoluble carrier on which a labeled antibody (or antigen) is immobilized; and a labeled antibody (or antigen) placed in the container. 3. An immunoassay method using the immunoassay device according to claim 1 or 2.