JPS63221246A - Dry type immunoassary element - Google Patents

Abstract

PURPOSE:To prevent reaction of labeling antigens and antibodies before a liquid to be inspected contacts a dry type analysis element for measuring enzyme activity consisting of a porous layer and reagent layer by incorporating the labeling antigens and antibodies dividedly in the respective layers of said element. CONSTITUTION:The reagent layer is integrated with the side opposite from the surface where the liquid of the porous layer is received. The respective layers have water penetratability and the antibodies to the non-labeling and labeling antigens among the component contributing to an antigen/antibody reaction are incorporated into the porous layer. On the other hand, the substantially all of the labeling antigens are incorporated into the reagent layer. The antibodies and the labeling antigens do not contact each other and do not react with each other before contact with the body fluid if the dry type analyzing element for measuring the enzyme activity constituted in such a manner is used and, therefore, the sensitivity of detection is enhanced.

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, 55-164356, and 59).
-102388), dry analytical elements generally measure the amount of reaction products or unreacted components of the reaction between the test component and the reagent contained within the analytical element by optical means, such as color development, discoloration,
Measure by spectrophotometry such as fluorescence and luminescence to 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 is a stack 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. The amount of antigen is measured as a decrease in fluorescence intensity using a competitive antigen-antibody reaction.

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 (the so-called B/F fraction M
), or an enzyme system whose activity changes between B and F 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.

[Configuration of the Invention] The first object of the present invention is to provide an integrated at least two
It consists of two water-permeable layers, one of which is a porous layer and the other is a reagent layer, the reagent layer being located on the opposite side of the porous layer to the liquid-receiving surface, and the antigen /A dry analytical element for enzyme activity measurement containing components involved in antibody reaction in a water-permeable layer, one of the components involved in the antigen/antibody reaction being a labeled antigen and the other being unlabeled and labeled. A dry analysis characterized in that the antibody is an antibody against an antigen, and substantially all of the antibody is contained in a porous layer and substantially all of the labeled antigen is contained in a reagent layer until it comes into contact with a test liquid. Achieved by elements.

The second of the above objects comprises at least two water-permeable layers integrated, having at least a water-permeable porous layer and a reagent layer, the reagent layer being a liquid-receiving surface of the porous layer. When producing a dry analytical element for measuring enzyme activity, which contains components involved in antigen/antibody reactions in the water-permeable layer, which is located on the opposite side, a porous layer is provided on the reagent layer containing the labeled antigen. This was achieved by coating or impregnating the porous layer with a composition that contains the antibody and does not dissolve or solubilize the labeled antigen in the reagent layer.

The second objective was also achieved by providing a porous layer pre-coated or impregnated with the composition containing the antibody on the reagent layer containing the labeled antigen when manufacturing the analytical element. .

In particular, after providing a reagent layer containing a labeled antigen and using a hydrophilic polymer as a binder by coating, etc., and providing a porous layer thereon,
One useful method is to apply or impregnate the porous layer with a composition that contains the antibody and does not swell the hydrophilic polymer in the reagent layer. As a composition that does not swell the hydrophilic polymer in the reagent layer, solutions of organic solvents such as alcohols, ethers, and ketones are useful. For example, ethanol, methanol, acetone, ethyl cellosolve, etc. can be used. .

Furthermore, it is also useful to adhere a porous layer containing an antibody by impregnating or coating it in advance onto a porous reagent layer containing a labeled antigen using a method such as that described in JP-A No. 61-4959. .

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

The porous layer in the present invention may be the top layer of the analytical element or a layer between the top layer and the reagent layer. spreading the liquid over an area approximately proportional to the amount of liquid supplied;
It may be a liquid spreading layer having a so-called metering effect (hereinafter sometimes referred to as a spreading layer), or it may be other than that. Membrane filter made of cloth made of natural fibers, cloth made of synthetic or semi-synthetic fibers, nonwoven fabric, paper, cellulose acetate, etc. which may be fibrous or non-fibrous - 1! ! It may be a combination of organic particles or organic particles, for example, the fibers described in JP-A-55-164356 and JP-A-60-222769. ! In addition to the quality layer, JP-A-49-53888, JP-A-58-70163
No. 61-4959, Japanese Patent Application No. 60-256408, No. 60-279859, No. 60-279860,
A porous layer such as that described in Japanese Patent No. 60-279861 is also suitable.

Various fluorescent substances can be used to fluorescently label an antigen. They can be classified according to their functional components, and for example, fluorescent substances with amino groups include 1-aminonaphthalene 2-aminonaphthalene p, p'-diaminostilbene 2-aminoacridine p, p'-diaminobenzophenone imine bis- 3-
Examples of phenolic fluorescent substances include amine pyridinium salts, indoles, 7-hydroxycoumarins, 3,6-hydroxyxanthenes, cervical sterophels, detracycline salicylic acid esters, etc.A typical example is fluorescein.

The antigen may be labeled with a luminescent substance, such as a chemiluminescent substance, e.g. 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 (G○[)), β-
D-galactosidase, peroxidase (POD>,
These include alkaline phosphatase (ALP), α-amylase, and luciferase.

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.: "Enzyme immunoassay (2nd edition) J (1982), edited by Tadashi Kawai: "Clinical Testing Techniques Complete Book 4 Immunology. Serum Test” (Igakushoin, published in 1977), pp. 97-102, B 1oehe+m, B 1
opl+ys, Res, coa++sun, , 74
, 538 (1977), C11nica Cl+i+*
ica Acta, 83, 161 (1978), etc. can be referred to.

Specific examples of rs antigens include fluorescein inthiocyanate tyroxine FITCT4゜GOD-binding human immunoglobulin (IgG), ALP-binding IgG, and PODP.
! A! Examples include α-fetoprotein.

The present invention can be applied to various known dry analysis elements. The element may have a multilayer configuration including a porous layer, a reagent layer, a support, a spreading layer, a detection layer, a light shielding layer, an adjacent filtration layer, a water absorption layer, a subbing layer, and other layers. A good example of such an analytical element is U.S. Pat. No. 3,992,158;
No. 4,042,335 and JP-A-55-16435
There is something disclosed in each specification of No. 6.

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) a second reagent layer, a light reflection layer, a first reagent layer on the support,
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 thereon in this order.

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, and (1) to 3 above. )
In (6), a filtration layer may be provided between the reagent layer and the detection layer or development layer.

In (3) to 7) above, a filtration layer may be further provided between the light reflection layer and the detection layer, the reagent layer or the spreading layer, between the reagent layer and the detection layer, or between the reagent layer and the spreading rmMI. In the case where the reagent layer is composed of multiple layers, a filtration layer may be further provided between the reagent layers.

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, examples include JP-A-58-70163, JP-A-61-4959, and JP-A-Sho 60. Porous layers such as those described in No. 256408, No. 60-279859, No. 60-279859, and No. 60-279861 are also suitable. Examples of Ni aqueous polymers include gelatin and derivatives thereof such as phthalated gelatin>, cellulose derivatives (rIA such as hydroxymethylcellulose), agarose, acrylamide polymers, methacrylamide polymers, acrylamide or methacrylamide in combination with various vinyl monomers, etc. Polymers etc. can be used.

The reagent layer can contain a substrate for the enzyme, an oxidizing agent, a coupler, an H buffering agent, 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
Good's as described in cbemisLry magazine Vol. 5 No. 2, pages 467 to 477 (1966)! 8 agents can be mentioned.

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 +*1sLry Magazine Volume 5.

When a porous layer is used as a spreading layer, it is preferably a layer that has a liquid metering effect. This is an effect of spreading the II sample in a substantially constant amount per unit area in the plane direction without substantially unevenly distributing the components contained therein.

As materials constituting the spreading layer and other porous layers, paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, tricot #i), glass fiber r paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferable as the developing layer, and woven fabrics may be treated with glow release as described in JP-A No. 57-66359. In order to adjust the RR concave area development speed etc. in the development layer, 1ki Kaisho 6
No. 0-222770, Patent Application No. 122875-1987, 6
1-122876, 6]--143754 may be contained.

An adhesive layer for adhering and laminating the porous layer may be provided on layers such as a reagent layer, a light guide layer, an 81 superlayer, a water absorption layer, a detection layer, etc. The adhesive layer is moistened with water for 15 minutes. It is preferably composed of a hydrophilic polymer, such as gelatin, gelatin derivatives, polyacrylamide, starch, etc., to which the porous layer can sometimes be adhered.

The light-shielding 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 light-reflecting layer or background layer.

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles such as titanium oxide and barium sulfate are dispersed using a hydrophilic polymer having film-forming ability as a binder. As the binder, gelatin, gelatin derivatives, polyacrylamide, etc. are preferable. For analytical elements, instead of providing a light-shielding layer, or at the same time, light-reflecting particles such as titanium oxide may be applied to the spreading layer, reagent layer, detection layer, etc. It may be included.

Example C] (Multilayer immune-integrated element for 74 measurements) [1]
Synthesis of fluorescein-labeled thyroxine JP-A-59-17
Fluorescein-labeled thyroxine (hereinafter referred to as FITC-T) was synthesized by the method described in 0768.

[2] Preparation of anti-thyroxine antiserum dispersion A liquid with the following formulation was dispersed for 1 hour using a homogenizer to prepare an anti-thyroxine antiserum dispersion.

Anti-thyroxine antiserum 1i+1 (as lyophilized powder) Nonylphenoxypolyethoxyethanol (*) 0.2H acetone
100g* rl = 15 C3) Multi-R sheet Tonolil:vA Aqueous solution of the following composition (a) is dried on a 180μ thick colorless transparent smooth film of polyethylene terephthalate coated with gelatin so that the thickness after drying becomes 2μl. (Reagent layer) (a) Gelatin
10g Water 190g Formalin 0.2FI FITC-7, 120μg Furthermore, gelatin was coated on the above reagent layer so that the thickness after drying was about 2μl to form a timing layer.

After wetting the timing layer with about 30 g/z” of water,
Polyethylene terephthalate spun paper (36 gauge, 50
The tricot knitted fabric consisting of D) was pressed and dried to form a spread layer.

Apply 120 c of a coating solution having the following composition (L+) to the above development layer.
It was coated at a ratio of "c/x" and dried to produce a TJI constant m integrated multilayer analytical element (1).

<b> Anti-thyroxine antiserum dispersion 5N polyvinylpyrrolidone 25g Nonylphenoxypolyethoxyethanol (*) 0.21FI methyl alcohol 500d *n=15 Also, except that the composition of the coating liquid (b) was changed to the following (c) In exactly the same way, integrated multilayer analysis element for T and i running (2)
was created.

(C) Anti-thyroxine antiserum dispersion 5z/Polyvinyl and Lolidone 25y Nonylphenoxypolyethoxyethanol (*) 0.2xfl water
500zf [4] Thyroxine (T,) measurement Serum was treated with activated charcoal to obtain T4-free serum, and a known amount of thyroxine was added to this to obtain thyroxine (T,) at various concentrations for calibration. Serum 10μ! to 0.3M glycine'-N a CR-N a OH1
20 μl of a mixture of a 15 μm bomb and 5 μl of a 0.1 M aqueous solution of a blocker reagent (sodium dodecylnaphthalesulfonate) was dropped onto the developing layer of each multilayer analytical element.

After standing at 25°C for 30 minutes, reflected fluorescence was measured from the polyethylene terephthalate film side. 0 Reflected fluorescence was measured using Hitachi Fluorescence Zt 650-10 (S) at an excitation wavelength of 495 nm and a fluorescence wavelength of 525 nm. carried out.

The obtained calibration curve is as shown in FIG.

It can be seen from FIG. 1 that analytical element (1) has significantly better performance than analytical element (2).

[Brief explanation of the drawing]

FIG. 1 is a calibration line of T obtained by fluorescence measurement of the immunoassay element of Example 1. Applicant Fuji Photo Film Co., Ltd. Figure 1 T4 (μg/jf) Procedural amendment March 25, 1988

Claims (1)

[Scope of Claims] 1) Consisting of at least two water-permeable layers integrated, one of which is a porous layer and the other a reagent layer, the reagent layer being a liquid permeable layer in the porous layer. The dry immunoassay element is located on the opposite side to the surface that receives the antigen and contains components involved in the antigen/antibody reaction in a water-permeable layer, and one of the components involved in the antigen/antibody reaction is a label. antigen,
The other is antibodies against unlabeled and labeled antigens,
A dry immunoassay element, wherein substantially all of the antibody is contained in a porous layer, and substantially all of the labeled antigen is contained in a reagent layer. 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 and the other a reagent layer, the reagent layer being opposite to the liquid-receiving side of the porous layer. A method for producing a dry immunoassay element in which a water-permeable layer contains a component that is located on the side and is involved in an antigen/antibody reaction, the method comprising: providing a porous layer on the reagent layer containing the labeled antigen; ,
A method comprising applying or impregnating a porous layer with a composition that includes the antibody and does not dissolve or solubilize the labeled antigen in the reagent layer. 4) The method according to claim 3), characterized in that the porous layer is a liquid spreading layer. 5) Consists of at least two integrated water-permeable layers, one of which is a porous layer and the other a reagent layer, the reagent layer being opposite to the liquid-receiving side of the porous layer. A method for producing a dry immunoassay element comprising a component involved in an antigen/antibody reaction in a water-permeable layer located on the side, the composition comprising the antibody being placed on the reagent layer comprising the labeled antigen. A method characterized by providing a porous layer pre-coated with or impregnated with. 6) The method according to claim 5), wherein the porous layer is a liquid spreading layer.