JPH01227964A - Production of immunological analysis element - Google Patents

Abstract

PURPOSE:To obtain a dry process immunological analysis element having high analysis sensitivity and accuracy by separately coating or impregnating a compsn. contg. an antigen-analogous body and a compsn. contg. a labeling body on or in a porous layer. CONSTITUTION:Bovine serum albumin (BSA) is dissolved in a buffer soln. and a soln. of thyroxine (T4) is added thereto, then a glutaraldehyde soln. is added thereto to effect reaction. The reaction liquid is dialyzed and is freeze-dried to obtain T4-BSA. The buffer soln. of the T4-BSA is impregnated into a nitrocellulose membrane filter and thereafter, an aq. soln. of the BSA is impregnated therein. The filter is cleaned with water after resting overnight and is dried at room temp. An ethanol soln. of a fluorescein isothiocyanate-labeled T4 antibody is impregnated in this antigen-immobilized film and is then rapidly dried. This film is partially adhered spottedly to a polyethylene terephthalate base and is cut. The cut film is sandwiched in a polyethylene frame provided with a window. The T4 measuring element is thus obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention is applicable to immunological analysis using antigen/antibody reactions.
The present invention relates to a method for manufacturing analytical elements for use.

[Prior Art] An analytical method for quantifying biochemical substances contained in body fluids using a dry analytical element is known.

In dry analytical elements, generally, the amount of reaction products or unreacted components of the reaction between the test component and the reagent contained in the analytical element is measured optically, such as by spectroscopy such as color development, color change, fluorescence, and luminescence. Measure by photometry 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.

In immunology, the antigen-antibody reaction is a reaction that specifically reacts and binds only to mutually corresponding antigens or antibodies, and is widely used for diagnosing autoimmune diseases and detecting trace substances in living organisms. 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 consisting of a porous medium, and a reaction layer containing immobilized antibodies on top. The amount of antigen is measured as a decrease in fluorescence intensity using a competitive antigen-antibody reaction between the antigen and the antigen.

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 analog to create an enzyme-labeled antigen, and the effect of the antigen on the enzyme activity is determined by 1111 determination in the sample solution. This is a method of analyzing antigens. In this method, the antigen-bound and enzyme-labeled antigen (denoted by B) and the unbound enzyme-labeled antigen (denoted by F) are separated (into so-called B/F fractions), or 13 and 1. What was needed was an enzyme system whose activity varied depending on the situation.

In order to establish such a system, two components are essential: an antibody, an enzyme, an antigen, or an antibody label and an antigen analog. If these two components are not prevented from reacting with each other before contact with the specimen, the detection rate of 15 degrees (shingetsu/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.

[1st aspect of the invention] [1st aspect of the present invention] The first aspect of the present invention is to provide a method for producing a dry immunoanalytical element having high analytical sensitivity and quantitative analysis accuracy.

[Structure of the Invention] The above-mentioned object of the present invention is to have at least 1-1) water-permeable porous layer, and contain an antigen analog and a labeled antibody as components involved in an antigen/antibody reaction in the porous layer. This was achieved by separately coating or impregnating a porous layer with a composition containing the antigen analog and a composition containing the labeled antibody. Preferably, the two compositions each consist of solvents with different polarities (eg, water and ethyl alcohol).

In particular, after applying or impregnating a first composition containing either an antigen analog or the labeled antibody, a second composition containing the other one and consisting of a solvent that does not swell the hydrophilic polymer is applied to a porous layer. One of the useful methods is to apply or impregnate. As a solvent that does not swell hydrophilic polymers,
Organic solvents such as lower alcohols (eg methyl alcohol, ethyl alcohol), ethers, ketones (eg acetone) are useful. The solvent used in the first composition is preferably water, but organic solvents such as those mentioned above may also be used.

An antigen analog refers to a substance that can bind to an antibody against the antigen in the same way as the antigen. Examples include a literal combination of an antigen and a protein, a physical combination of an antigen and latex, and a combination of an antigen and a solid support.

A porous expander pre-impregnated or coated with a composition containing an antigen analog and a composition containing the labeled antibody (one of which is an aqueous solution and the other of which is a solvent that does not swell the hydrophilic polymer). It is also a useful method to adhere the layer to another water-permeable layer, such as the soil of the reagent layer, as in JP-A-55-164356.

Furthermore, a porous layer containing a composition containing an antigen analog and a composition containing a labeled antibody by impregnation, coating, etc. in advance is placed on top of another porous layer (for example, a reagent layer for PA). A method of adhesion such as that disclosed in No. 61-4959 is also useful.

Alternatively, a porous layer (e.g., a spreading layer) pre-impregnated or coated with a first composition (e.g., an aqueous solution) containing an antigen analog may be placed on top of another water-permeable layer (e.g., a reagent layer). After adhesion by a method such as, a second composition containing a labeled antibody may be applied to the porous layer.

Conversely, impregnating a porous layer with a composition containing a labeled antibody,
After adhering to the pond layer, a composition containing an antigen analog may be applied.

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 uppermost layer of the analytical element, or may be a layer between the uppermost layer and the reagent layer, and spreads the liquid over an area approximately proportional to the amount of liquid supplied.
A fabric made of natural fibers, which may be a liquid spreading layer having a so-called metering effect (hereinafter sometimes referred to as the spreading layer) or may be other than that, and may be fibrous or non-fibrous. Membrane filter made of cloth, nonwoven fabric, paper, cellulose acetate, etc. made of synthetic or semi-synthetic fibers 1 Any combination of inorganic or organic particles may be used. For example, JP-A-55-164356, JP-A-60-22
In addition to the fibrous layer described in No. 2769, etc., JP-A-49
-53888, JP-A No. 58-70163, JP-A No. 61-
No. 4959, Patent Application No. 60-256408, No. 60-2
No. 79859, No. 60-279860, No. 60-27
Porous layers such as those described in US Pat. No. 9,861 are also suitable.

Various fluorescent substances can be used to fluorescently label antibodies. They can be classified according to their functional components, and for example, fluorescent substances with amine groups include 1-aminonaphthalene 2-aminonaphthalene p, p'-diaminosdilbene 2-aminoacridine p, and p'-diaminobenzophenonimine. Bis-3-
Aminopyridinium salts Indoles Examples of the phenolic fluorescent substances include 7-hydroxycoumarins, 3,6-dihydroxyxanthenes, tetracycline salicylic acid esters, etc. A typical example is fluorescein.

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

When enzymatically labeling antibodies, 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. Typical examples include glucose oxidase (COD), β-
D-galactosidase, peroxidase (POD),
These include alkaline phosphatase (ALP), α-amylase, and luciferase.

Regarding the method of binding a label to an antibody and the labeled antibody that can be used in the present invention, please refer to Eiji Ishikawa et al., "Enzyme immunoassay (2nd edition) J (1982)," Tadashi Kawai, II: "Clinical Test Techniques 4 Immunology." Serum Test” (Igakushoin, published in 1977), pp. 97-102, B 1ocl+em, I3
1opl+ys, Res, Coaemun,
, 74.538 (1977), Clnica Ch.
Reference may be made to the descriptions in Imica Acta, 83, 161 (1978) and the like.

Specific examples of labeled antibodies include fluorescein isothiocyanate (FrTC)-conjugated thyroxine (T) antibody, GOD
Examples include conjugated anti-human immunoglobulin (IgG), ALP-conjugated anti-IgG, and POD-labeled anti-α-fetoprotein.

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

The detection system for the substrate and the enzyme reaction product may be present in the same layer or in different layers. No particular object detection system is required.

The present invention can be applied to various known dry analysis elements. The elements include a porous layer, a reagent layer, a support, and a spreading layer.
J1! ! , a detection layer, a light-shielding layer, an adhesive layer, a filtration layer, a water-absorbing layer, a subbing layer, and other layers.
No. 158, No. 4,042,335 and JP-A-55-
There are some disclosed in each specification of No. 164356.

When using a light-transparent water-impermeable support, practical configurations of the dry analytical element of the present invention include (1) a reagent layer on the support and a spreading rM 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) One that has a detection layer, a light reflection layer, a reagent layer, and a development layer on the support.

(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 in this order.

The detection layer is generally a layer in which a dye generated in the presence of a test component diffuses and can be optically detected through a light-transmitting support, and is a layer that can be composed of a hydrophilic polymer and contains a mordant for the dye, such as an anion. A cationic polymer may also be included for the coloring matter.

The water-absorbing layer generally refers to a layer in which a dye generated in the presence of a test component does not substantially diffuse, and can be composed of a hydrophilic polymer that easily swells.

The layer containing the antigen analog and labeled antibody may be a spreading layer or a reagent layer.

When using an enzyme-labeled antibody, the reagent composition for detecting the enzyme reaction product may be contained in the layer containing the antigen analog and the labeled antibody, but it may also be contained in a separate non-porous or porous layer. You may let them. 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 between the reagent layer and the support. Alternatively, the composition for producing the intermediate may be contained in a layer farther from the support than the reagent layer, for example, the spreading fm layer, and the indicator may be contained in the reagent layer.

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 the detection layer. In (1) or 3) and (6) above, a filtration layer may be provided between the reagent layer and the detection layer or the development layer. Good, above (3) or 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. In this case, a filtration layer may be further provided between the reagent layers.

A preferred material for the light-transparent, water-impermeable support is polyethylene terephthalate. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is applied.

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. 60-279860, No. 60-279861 are also suitable. Examples of FA aqueous polymers include gelatin and derivatives thereof (e.g. phthalated gelatin), cellulose derivatives (e.g. hydroxymethylcellulose), agarose, acrylamide polymers, methacrylamide polymers, copolymers of acrylamide or methacrylamide with various vinyl monomers. Combination 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 an enzyme-labeled antibody is included as a labeled antibody, a substrate for the enzyme is essential.

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 r-Proteins and Enzymes 1 (Takeshi Horio et al., Nankodo, 1981), the above-mentioned Biocl+
The selection can be made by referring to literature such as volume 5 of emistry magazine.

When a porous layer is used as a spreading layer, it is preferable that the layer has a liquid metering function.The liquid metering function refers to a layer that collects the components contained therein by dropping a liquid sample onto its surface. This is an effect of spreading the particles in the direction of the surface at a rate of a substantially constant amount per unit area without substantially unevenly distributing the particles.

As materials constituting the expanded T7F1 layer and other porous layers, P paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, tricot fabric), glass fiber 2 paper, etc. can be used. Among these, textiles,
Knitted fabrics are preferred. Woven fabrics may be subjected to glow discharge treatment as described in JP-A No. 57-66359. For the spreading layer, in order to adjust the development surface finish, development speed, etc., No. 60-222770, patent application No. 122875-1987,
It may contain a hydrophilic polymer or a surfactant as described in Nos. 61-122876 and 61-143754.

An adhesive layer for adhering and top layering the porous layer may be provided on layers such as a reagent layer, a light guide layer, a filtration layer, a water absorption layer, a detection layer, etc. When the adhesive layer is swollen with water, Preferably, it consists of a hydrophilic polymer to which the porous layer can be adhered, such as gelatin, gelatin derivatives, polyacrylamide, starch, etc.

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,
Particularly 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 a film-forming ability as a binder.The binder is gelatin or a gelatin derivative. , polyacrylamide, and the like, light-reflecting particles such as titanium oxide may be included in the developing layer, reagent layer, detection layer, etc. instead of or simultaneously with the light-shielding layer.

[Example] (1) Synthesis of T, -BSA 15 mg of bovine serum albumin (BSA) was dissolved in 3 ml of phosphate MHIt solution (pH 8, 0, 0, 1 M), and 3 ml of T, solution (thyroxine 10 mg) To this solution, 0.2 ml of 1% geltaraldehyde solution was slowly added and reacted at 4° C. for 3 hours. The reaction solution was dialyzed for 2 days and lyophilized to obtain T4-BSA.

(2) Preparation of antigen-immobilized membrane A nitrocellulose membrane filter (manufactured by Millibore) was impregnated with the following solution (λ), left overnight, and then thoroughly washed with water.

(A) 74 BSA (above) 15 μg carbonate buffer (pH 9) 10 companies Next, add 300 m of BSA to this membrane filter.
The membrane was impregnated with a solution of Loaf g dissolved in water Loaf, left overnight, thoroughly washed with water, and dried at room temperature to obtain an antigen-immobilized membrane.

(3) Preparation of T4 measurement element After impregnating the antigen-immobilized membrane with a solution having the following composition (C),
Dry quickly (FITC means fluorescein isothiocyanate).

(C) FITC-labeled anti-labeled antibody solution 1ml (contains 40μg of antibody dry powder) Ethanol 9 Ugly! Next, this was placed on a PET support with a thickness of 180μ.
-138756 The dotted portions were adhered by the method described in Example 3. Cut into 15mm squares, top and bottom diameter 10g+
- Insert into a polystyrene frame with a window, T
, was defined as the measurement element [1].

On the other hand, the composition (C) of the solution to be impregnated into the antigen-immobilized membrane is as follows (
A comparative T4 measurement element [2] was prepared in the same manner as the above T measurement element except for changing to D).

(D) FITC-labeled anti-labeled antibody 4 anti (Contains 40 μg of antibody dry powder) Distilled water 9 brass! (4) Measurement of thyroxine A known amount of T in serum that does not contain thyroxine (T,).

0.3M glycine M was added to 5 μl of serum containing T.
Added 5 μl of buffer solution (pH 9.0, containing 0.05 MF sodium decylnaphthalene sulfonate) and spotted (dropped) it on each analytical element. After leaving it at 37°C for 15 minutes,
0.3M glycine M buffer for each analytical element (same as above)
The operation of dropping 15 μl and wiping was repeated twice to wash away free FITC-labeled antibody.

Then, use a fluorometer (Hitachi model 650-10 (
S)), excitation wavelength 495ns, fluorescence wavelength 525n
Fluorescence was measured from the PET support side. The results obtained are shown in FIG.

As is clear from FIG. 1, the analytical element [1] of the present invention exhibited superior immunoassay properties compared to the comparative analytical element [2].

[Brief explanation of the drawing]

FIG. 1 is a graph showing the immunoassay characteristics of the analytical element for T4 measurement. Applicant: Fuji Photo Film Co., Ltd.

Claims (3)

[Claims] (1) A method for producing a dry immunoassay element, which has at least one water-permeable porous layer and contains an antigen analog and a labeled antibody in the porous layer as components involved in antigen/antibody reactions. A method for producing a dry analytical element, comprising separately applying or impregnating a composition containing the antigen analog and a composition containing the labeled antibody onto the porous layer. (2) In claim (1), after coating or impregnating the porous layer with the composition containing the antigen analogue, a composition containing the labeled antibody and comprising a solvent that does not swell the hydrophilic polymer is applied to the porous layer. A method characterized by coating or impregnating a porous layer. (3) In claim (1), after applying or impregnating the composition containing the labeled antibody, a composition containing the antigen analog and comprising a solvent that does not swell the hydrophilic polymer is applied to the porous layer. A method characterized by coating or impregnating.