JPH01237455A - Dry immunity analyzing element - Google Patents

Abstract

PURPOSE:To obtain high analyzing sensitivity and accuracy in quantitative analysis, by a constitution wherein at least two water permeability layers form a unitary body, at least one of the water permeability layers is a porous layer, and components related to the reaction of antigen/antibody are added in the water permeability layers. CONSTITUTION:An element comprises at least two water permeability layers which form a unitary body. At least one of the water permeability layers is a porous layer. Components related to the reaction of antigen/antibody are added in the water permeability layers. One of the components related to the antigen/antibody is the labelled antigen. The other is the antibody for the non- labelled antigen and the labelled antigen. The labelled antigen is incorporated in the porous layer until the time when liquid to be checked comes to contact. The porous layer does not have the antibody substantially. The antibody is incorporated in the second water permeability layer which is located at a position separated remotely from the surface that receives liquid of the analyzing element from the porous layer. Said second layer does not incorporate the labelled antigen substantially. In this way, high analyzing sensitivity and the accuracy in quantitative analysis can be maintained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to analytical elements useful for immunological analysis utilizing antigen/antibody reactions.

[Prior Art] Analytical methods for quantifying biochemical substances contained in body fluids using dry analytical elements are known (for example, Japanese Patent Application Laid-open Nos. 49-53888, 55-1, 64356, and 5).
No. 9-102388). In dry analytical elements, the amount of reaction products or unreacted components of the reaction between the test component and the reagent contained in the analytical element is generally measured optically, for example, by spectrophotometry such as color development, color change, fluorescence, and luminescence. and quantify the test component. By using a dry analytical element, it is possible to analyze a specific component, such as a biochemically active substance, in a liquid simply, quickly, and with high precision.

Immunological analysis using dry analytical elements is also known, for example, as described in JP-A-49-53888, JP-A-59-102388, and US Pat. No. 4,459.358.

Antigen-antibody reactions in immunology are reactions that specifically react and bind only mutually corresponding antigens or antibodies, and are widely used for diagnosis of autoimmune diseases, detection of trace substances in living organisms, etc. However, since it requires skill to operate, a method that can perform accurate measurements with simple operations is desired for clinical testing.

One typical method of simple antigen amount measurement using a multilayer analytical element is disclosed in Japanese Patent Application Laid-Open No. 77356/1983. The analytical element used in this method consists of a development layer containing a fluorescently labeled antigen, a distribution layer made of a porous medium, and a reaction layer containing an immobilized antibody. Utilizing a competitive antigen-antibody reaction between antigens, the amount of antigen is measured as a decrease in fluorescence intensity.

Enzyme immunoassay (EIA) is also considered as an immunoassay method with relatively high detection sensitivity. A typical method is known from Japanese Patent Publication No. 53-27763, in which an enzyme is bound to an antigen or its derivative to create an enzyme-labeled antigen, and the antigen in a test solution is analyzed by measuring the effect of the antigen on enzyme activity. This is the way to do it. In this method, the enzyme-labeled antigen bound to the antibody (denoted by B) and the unbound enzyme-labeled antigen (denoted by F) are separated (so-called B/F separation), or the activity of B and F is separated. A variable enzyme system is required.

In order to establish these systems, two components are essential: an antibody and a labeled antigen, or an antibody label and an antigen derivative, and care must be taken to ensure that these two components do not react with each other before contact with the sample. If this is not done, the detection sensitivity (signal/background ratio) will drop significantly. Many of the dry analytical elements known so far have not been able to obtain satisfactory analytical sensitivity due to this factor.

[Object of the Invention] An object of the present invention is to provide a dry immunological analytical element and a method for producing the same, which have high analytical sensitivity and quantitative analytical accuracy.

[Structure of the Invention] The first object of the present invention is to provide an integrated at least two
A dry immunoassay element comprising two water permeable layers, at least one of the water permeable layers being a porous layer, and the water permeable layer containing a component involved in an antigen/antibody reaction. One of the components involved in the antibody reaction is the labeled antigen, and the other is antibodies against unlabeled and labeled antigens.Until the test liquid comes into contact with the sample, the labeled antigen is contained in the porous layer The layer is substantially free of antibody, said antibody being contained in a second water-permeable layer located further from the liquid-receiving surface of the analytical element than said porous layer, and said layer being substantially free of labeled antigen. This was achieved using a dry analytical element that is characterized by not containing

The second of the above objects consists of at least two integrated water-permeable layers, one of which is a porous layer, and the antigen/
When manufacturing a dry immunoassay element that contains a labeled antigen and antibodies against unlabeled and labeled antigens in a water-permeable layer as components involved in antibody reactions, a porous layer is provided on the water-permeable layer containing the antibody. This was achieved by applying or impregnating the porous layer with a composition that contains the labeled antigen and does not dissolve or solubilize the antibody in the water-permeable layer.

The second of the above objectives can also be achieved by providing a porous layer coated or impregnated with the composition containing the labeled antigen in advance on the water-permeable layer containing the antibody when manufacturing the analytical element. It was done.

In particular, after providing a water-permeable layer containing an antibody and using a hydrophilic polymer as a binder by coating, etc., and providing a porous layer on top of the water-permeable layer,
One useful method is to apply or impregnate the porous layer with a composition that contains the labeled antigen and does not swell the hydrophilic polymer. Examples of compositions that do not swell hydrophilic polymers include lower alcohols, ethers, ketones,
Solutions of organic solvents such as low boiling esters are useful.

For example, ethanol, methanol, acetone, ethyl cellosolve, etc. can be used.

In addition, on the porous layer containing the antibody, a labeled antigen is pre-impregnated.
For example, a porous layer contained by coating etc.
A method of adhesion such as that disclosed in No. 4959 is also useful.

Known methods can be used for impregnating or coating the porous layer. For example, dip coating, doctor coating, hopper coating, curtain coating, etc. are selected and used as appropriate.

The porous layer in the present invention may be the top layer of the analytical element, or it may be a layer between the top layer and another water-permeable layer. A liquid spreading layer (hereinafter referred to as "liquid spreading layer") has a so-called metering effect, spreading liquid over an area approximately proportional to the amount of liquid supplied.
(sometimes referred to as a deployment layer) or other layers. It may be fibrous or non-fibrous. Membrane filter 1 made of natural fibers, synthetic or semi-synthetic fibers, nonwoven fabrics, paper, cellulose acetate, etc. Any combination of inorganic or organic particles may be used. For example, in addition to the fibrous layer described in JP-A-55-184356 and JP-A-60-222769,
JP 49-53888, JP 58-70163, JP 61-4959, JP 60-256408,
Porous layers such as those described in No. 60-279859, No. 60-279860, and No. 60-279861 are also suitable.

Various fluorescent substances can be used to fluorescently label antigens. They can be classified according to their functional components, and for example, fluorescent substances with amine groups include 1-aminonaphthalene 2-aminonaphthalene p, p'-diaminostilbene 2-aminoacridine p, p'-diaminobenzophenone imine bis- 3-
Examples of the aminopyridinium salt indole phenolic fluorescent substances include 7-hydroxycoumarins, 3,6-dihydroxyxanthenes, tetracycline salicylic acid esters, and the like. A typical example is fluorescein.

The antigen may be labeled with a luminescent substance, such as a chemiluminescent substance. For example, 2) 3-dihydro-1,4-phthalazinedione, typified by luminol, or 2.4.5-
) Riphenylimidazoles can be used.

When enzymatically labeling an antigen, various enzymes can be used. Enzymes are selected taking into consideration the ability to obtain sufficiently high enzyme activity, the stability of the enzyme, the influence of binding on enzyme activity, and the like. It can be selected from those listed in Tables 1 and 2 of the specification of Japanese Patent Application Publication No. 56-500901. Representative ones are glucose oxidase (COD), β-D
- galactosidase, peroxidase (POD), alkaline phosphatase (ALP), α-amylase, luciferase, etc.

Regarding the method of binding a label to an antigen and the labeled antigen that can be used in the present invention, please refer to Eiji Ishikawa et al. Serum Test” (Igakushoin, published in 1977), pp. 97-102, Biochem, B 1op
hys, Res, commun,, 74,538 (19
77), Clnica Chimica Acta
, 83, 161 (1978) and the like.

Specific examples of labeled antigens include fluorescein isothiocyanate-conjugated thyroxine FITC-T4°GOD-conjugated human immunoglobulin (IgG), AIP-conjugated IgG,
Examples include POD-labeled α-fetoprotein.

The present invention can be applied to various known dry analysis elements. In addition to the porous layer and the reagent layer, the element may have a multilayer configuration including a support, a spreading layer, a detection layer, a light shielding layer, an adhesive layer, a filtration layer, a water absorption layer, a subbing layer, and other layers. . As such an analytical element, for example, U.S. Patent No. 3,992,15
No. 8, No. 4,042,335, JP-A-55-1.64
No. 356, JP-A-62-138756, JP-A No. 62-13
There are those disclosed in the specifications of No. 8757 and No. 62-138758.

When using a light-transmitting and water-impermeable support, the practical configuration of the dry analytical element of the present invention is (1) having a reagent layer on the support and a developing layer thereon.

(2) A device having a detection layer, a reagent layer, and a developing layer in this order on a support.

(3) One having a reagent layer, a light reflecting layer, and a developing layer in this order on a support.

(4) A support having a detection layer, a reagent layer, a light reflection layer, and a development layer in this order.

(5) A support having a detection layer, a light reflection layer, a reagent layer, and a development layer in this order.

6) On the support, a second reagent layer, a light reflection layer, a first reagent layer,
It has development layers in this order.

(7) A support having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and a developing layer in this order.

The detection layer is generally a layer in which a dye or the like generated in the presence of a test component can be diffused and optically detected through a light-transmitting support, and can be composed of a hydrophilic polymer. Mordants for dyes may be included, such as cationic polymers for anionic dyes.

In (1) to 5) above, the reagent layer may consist of a plurality of different layers. A water absorption layer may be provided between the support and the reagent layer or detection layer. The water-absorbing layer generally refers to a layer in which the dye produced in the presence of the test component does not substantially diffuse, and can be composed of a hydrophilic polymer that easily swells.

In Section 2 (1) to 3) and (6), a filtration layer may be provided between the reagent layer and the detection layer or development layer. the above(
In 3) to 7), a filtration layer may be further provided between the light reflection layer and the detection layer, the reagent layer, or the development layer, between the reagent layer and the detection layer, or between the reagent layer and the development layer. When the reagent layer consists of multiple layers, a filtration layer may be provided between the reagent layers.

When using an enzyme-labeled antigen, it is necessary to measure enzyme activity in order to measure the results of the antigen/antibody reaction, and a detection system for enzyme substrates and enzyme reaction products is required. Although the substrate may be present in the same layer as the labeled antigen, it is preferably contained in a different layer. It is preferred that at least one of the substrate or the enzyme-labeled antigen is coated or impregnated with a solvent in which the enzymatic reaction of the enzyme-labeled antigen does not substantially occur.

The substrate and the enzyme reaction product detection system may be present in the same layer or may be contained in different layers. When the substrate is a self-developing substrate, a detection system for the enzyme reaction product is not particularly required.

When using an enzyme-labeled antigen, the reagent composition for detecting the enzyme reaction product may be contained in the layer containing the labeled antigen, but may also be contained in a separate non-porous layer or porous layer. . For example, a composition that produces an intermediate by reaction with an enzyme reaction product is placed in the first reagent layer, and a composition (indicator) that can react with the intermediate to produce a dye or the like is placed in the first reagent layer and the support. It may be included in the second reagent layer between. In addition, a layer further from the support than the reagent layer contains the composition for producing the intermediate;
For example, it may be contained in a light reflective layer, etc., and an indicator may be contained in a reagent layer.

As the reagent layer of the dry analytical element of the present invention, in addition to a substantially uniform layer containing a hydrophilic polymer as a binder, for example, JP-A-58-70163, JP-A-61,-4959, JP-A-Sho. No. 62'-11,6258, JP-A-62-1.28
No. 756, No. 62-1.38757, No. 621.387
Porous layers such as those described in No. 58 and others are also suitable. Examples of hydrophilic polymers include gelatin and derivatives thereof such as phthalated gelatin, cellulose derivatives (
For example, hydroxyethyl cellulose), agarose, an acrylamide polymer, a methacrylamide polymer, a copolymer of acrylamide or methacrylamide with various vinyl monomers, etc. can be used.

The reagent layer can contain a substrate for the enzyme, an oxidizing agent, a coupler, a buffer, etc., if necessary. When the reagent layer contains an enzyme-labeled antigen, a substrate for the enzyme is required.

Examples of buffers that can be contained in the reagent layer of the analytical element of the present invention include carbonates, borates, phosphates, and
Examples include the buffering agent of Good, which is described in Chemistry Magazine, Vol. 5, No. 2, pages 467 to 477 (1966).

These buffers are described in Basic Experimental Methods for Proteins and Enzymes J (Takeshi Horio et al., Nankodo, 1981), the above-mentioned Bioche
The selection can be made by referring to literature such as Mistry magazine Vol. 5.

When a porous layer is used as a spreading layer, it is preferably a layer that has a liquid metering function. Liquid metering action is an action that spreads a liquid sample dotted onto the surface at an approximately constant rate per unit area in the direction of the surface without substantially unevenly distributing the components contained therein. It is.

As the material constituting the spreading layer and other porous layers, filter paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, tricot fabric), glass fiber filter paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferable as the spreading layer. Fabrics and the like may be subjected to glow discharge treatment as described in JP-A No. 57-66359. For the development layer, in order to adjust the development area, development speed, etc.,
No. 222770, Patent Application No. 61-122875, 61-
It may contain a hydrophilic polymer or a surfactant as described in Nos. 122876 and 61-143754.

An adhesive layer for adhering and laminating the porous layer may be provided on layers such as a reagent layer, a light guide layer, a permeation layer, a water absorption layer, and a detection layer. Preferably, the adhesive layer comprises a hydrophilic polymer, such as gelatin, gelatin derivatives, polyacrylamide, starch, etc., which is capable of adhering the porous layer when swollen with water.

The light-reflecting layer is used to measure the detectable changes (color change, color development, etc.) that occur in the detection layer, reagent layer, etc. from the light-transmitting support side. liquid color,
In particular, when the sample is whole blood, it blocks the red color of hemoglobin and functions as a background layer. As the light-reflecting layer, a water-permeable layer in which light-reflecting fine particles such as titanium oxide or barium sulfate are dispersed using a hydrophilic polymer as a binder is suitable. Preferable binders include gelatin, gelatin derivatives, polyacrylamide, and the like. In the analytical element, instead of providing a light-reflecting layer, or at the same time, light-reflecting particles such as titanium oxide may be included in the developing layer, reagent layer, detection layer, etc.

[Example 1] (1) Synthesis of fluorescein-labeled thyroxine
-170768, fluorescein-labeled thyroxine (hereinafter abbreviated as FITCT4) was synthesized.

(2) Preparation of antiserum-containing sheet An aqueous solution of the following composition (a) is applied onto a 180 μm thick colorless transparent smooth film of polyethylene terephthalate that is undercoated with gelatin so that the thickness after drying is 2 μm, and then dried. Water absorption layer) (a) Gelatin
10g water 190I? Formalin 0.2 g After the water absorption layer was moistened with 20 g/x2 of water, a microfilter FM300 (manufactured by Fuji Photo Film Co., Ltd.) was crimped onto it.

Add 100CC/II2 of the coating liquid of the following composition (b) to this.
The antiserum-containing sheet [1] was obtained by coating and drying at a ratio of

(b) Anti-thyroxine antiserum nonylphenoxy polyethoxyethanol 0.041f water 10C1+y(3)
Preparation of FITC-T4-containing sheet An I-ricott knitted fabric made of polyethylene terephthalate spun yarn (36 gauge, 50D) was immersed in a liquid having the following composition (C) and then dried.
A sheet containing TC-T4 was prepared (FITC-T means fluorescein isothiocyanate-bonded thyroxine).

(C) FITC-T, 24μs polyvinylpyrrolidone 5I? Nonylphenoxypolyethoxyethanol 0,1 *g water
200 companies (4) Preparation of multilayer sheet The above antiserum-containing sheet and FITC-T-containing sheet were laminated by dot adhesion according to the method described in JP-A-62-138756 to form a multilayer sheet for measuring thyroxine (T4). .

(5) Measurement of thyroxine (T4) Serum was treated with activated charcoal to obtain T-free serum, and a known amount of thyroxine was added to this to obtain serum containing various concentrations of thyroxine (T4). 15μβ, 0.3M glycine-NaCf-NaOH buffer 7.5μβ and blocker reagent (sodium dodecylnaphthalene sulfonate)
30 μl of the mixture containing 7.5 μf of 01M aqueous solution was dropped onto the multilayer analytical element.

After standing at 25° C. for 30 minutes, reflected fluorescence was measured from the support side. A fluorometer (Hitachi Model 650-10 (S)) was used to measure reflected fluorescence, with an excitation wavelength of 495 nm,
Measurements were performed at a fluorescence wavelength of 525 nm.

The obtained calibration curve is as shown in FIG.

[Example 2] (Preparation of theophylline immunoassay element and measurement method using the same) A multilayer immunoassay element was produced according to the following method.

=19- (1) Application of antibody 180 μI Apply a solution of the following composition N on a PET support at 90 μl.
After applying an amount equivalent to c/z2 and drying it, apply a composition powder on top of it at a rate of 100cc/w2 to form gelatin Sera 1.
In the cooled state, the glow discharge treated polyester knitted fabric was laminated from 1 to 1 and then dried.

Gelatin 25y surfactant
2g (Olin Surfactant 1
0G) Distilled water 444g Nitrotetrazolium blue (NTB) 3.2g Gelatin 20y Surfactant 2g (Olin Surfactant OG) Water 260g Citric acid 3g2O- (pH adjusted to 7.7) Diaphorase 1% solution 20y Next Theophylline antibody solution (rEMI manufactured by Cyba, USA)
5 of Reagent A) of TJ Theophylline 100 Assay Kit
% polyvinylpyrrolidone dissolved in distilled water (1211%) was applied to an area of 800 cm shoulder 2 and dried to serve as a base for a multilayer immunoassay element.

(2) Incorporation of labeled antigen A polyester knitted fabric subjected to glow discharge treatment was immersed in a solution having the following composition and dried to obtain a labeled antigen-containing sheet.

(3) Preparation of multilayer immunoassay element The undercoat (1) above and the labeled antigen-containing sheet (2) were laminated and adhered to form a multilayer immunoassay element.

(4) Calibration curve of theophylline using the above immunoassay element The immunoassay element prepared in (3) above was cut into 15 z squares, inserted into a plastic mount with holes of 10 z x diameter on the top and bottom, and theophylline was added at 0.2 sq. .5
, 5.10.20.A solution in which serum calibrator containing 40 μg/x1 (in the "Emit" kit manufactured by Cyba) and the buffer solution of the "Emit" theophylline kit manufactured by Cyba were mixed in a weight ratio of 1 = 4. 30μ each! The reflected absorbance change at a wavelength of 540 nm (
The difference between 5 minutes and 1 minute after application was measured. The results obtained are shown in FIG. It can be seen from FIG. 2 that a good calibration curve was obtained for the amount of theophylline in serum between 2.5 and 40 μg/xl.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a calibration curve for thyroxine, and FIG. 2 is a graph showing a calibration curve for theophylline. Applicant Fuji Photo Film Co., Ltd. Figure 1

Claims (1)

[Scope of Claims] 1) Consisting of at least two water-permeable layers integrated, at least one of the water-permeable layers being a porous layer;
A dry immunoassay element containing components involved in the antigen/antibody reaction in the water-permeable layer, one of the components involved in the antigen/antibody reaction being a labeled antigen,
The other one is an antibody against the unlabeled and labeled antigen, and the porous layer contains the labeled antigen but is substantially free of the antibody, and is further away from the liquid-receiving surface of the analytical element than the porous layer. A dry immunoassay element characterized in that the other water-permeable layer located at the top contains the antibody and substantially does not contain the labeled antigen. 2) An analytical element according to claim 1, characterized in that the porous layer is a liquid spreading layer. 3) Consists of at least two integrated water-permeable layers, one of which is a porous layer, containing labeled antigen and antibodies against unlabeled and labeled antigens as components involved in the antigen/antibody reaction. A method for producing a dry immunoassay element comprising a water-permeable layer containing a porous A method characterized in that, after providing the porous layer, the porous layer is coated or impregnated with a composition that contains the labeled antigen and does not dissolve or solubilize the antibody in the water-permeable layer. 4) Consisting of at least two integrated water-permeable layers, one of which is a porous layer, containing labeled antigen and antibodies against unlabeled and labeled antigens as components involved in the antigen/antibody reaction. A method for producing a dry immunoassay element comprising in a water-permeable layer, the water-permeable layer containing the antibody and located farther from the liquid-receiving surface of the analytical element than the porous layer; A method characterized by providing a porous layer coated or impregnated with a composition containing a labeled antigen in advance.