JPS5913956A - Material for immunoanalysis - Google Patents

Abstract

PURPOSE:To improve quantitative performance of immunoanalysis, by providing a developing layer which is easy to control a constant vacancy ratio, and which is composed of a particle-combined matter formed by particle structures having high adhesion strength. CONSTITUTION:An analytical element for analysing components in a fluid sample has at least one layer of fibrous structure, and at least one layer of particle- combined matter having a reactive group. The said layer of particle-combined matter is composed of a particle-combined matter of nonswelling and three- dimensional lattice which has mutually connected vacant voids at 25-85% vacancy ratio capable of transporting the fluid sample. The said particles are heat-stable organic polymer particles of 1-350mu size having a reactive group, and are not swelled or permeated by the said fluid sample. In the present invention, it is preferable that the polymer particles are either product by direct combination among the particle units through the said reactive group at their at their contact parts, or product by combination among the particle units with the intervention of low molecular compound through the said reactive group at their combining parts.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to analytical chemistry, and more particularly to an analytical element for analyzing all predetermined specific components in a fluid, and more particularly to quantitative analysis of all specific components in a biological fluid sample. Regarding elements.

Conventionally, many methods for analyzing components in fluid samples have been developed. An example is an automatic quantitative analyzer. These are particularly useful in clinical laboratories of hospitals.

Such an automatic analyzer is described, for example, in U.S. Pat. No. 2,797.
The sample, diluent, and analytical reagents are mixed together in a continuous flow analysis as described in No. 149, No. 149, and this? A method of transporting the sample to an analytical device is used.

However, such continuous automatic analyzers are complex and expensive, require skilled operating engineers, and require repeated cleaning operations after every analytical operation, which takes a great deal of time. However, these waste liquids inevitably cause environmental pollution problems.

On the other hand, in contrast to the above-mentioned analysis system that uses a solution, there is an analysis system that uses dry chemistry (Ir). These are called test strips or test strips, such as U.S. Pat.
As described in No. 050,575 or No. 5,061,525, an absorbent carrier such as a piece of paper is completely impregnated with an analytical reagent solution and provided in a dried form. This test piece is immersed in a fluid sample, which is the specimen, and then pulled out, and the change in color or concentration of the test piece is determined visually or with a device such as a densitometer.

These test pieces are useful because they are easy to handle and provide immediate results. However, these test strips comprising reagents supported in absorbent carriers have various serious drawbacks, which limit their use to qualitative or semi-quantitative analysis.

To overcome these drawbacks, U.S. Patent No. 5.992
, No. 158 was developed. This is a structure in which a reagent layer containing all analytical reagents and a diffusion layer made of a uniformly porous non-fibrous porous medium are laminated on a transparent support.

The above patent specification describes a porous film formed of silica gravel particles, white pigment, or inert white glass beads using a polymer material binder such as cellulose ester.

However, these films inherently have only weak strength and are highly susceptible to breakage, making it difficult to supply them stably, and when fluid samples containing cells such as blood cells or large complex proteins are applied. However, they have the drawback of causing undesirable phenomena such as pore clogging or non-uniform permeation. Also, from the manufacturing standpoint, it is necessary to strictly control the coating conditions, and if these conditions are deviated, it is difficult to obtain a constant porosity. Additionally, particulate materials such as microcrystalline colloidal particles, ie, cellulose microcrystals, tend to swell in the presence of aqueous fluid samples. therefore,
Porous granular structured layers produced from the above-mentioned materials contain the drawback that upon application of a fluid sample, the voids within the layer are partially or completely occluded, significantly impeding fluid flow. In another aspect of the same patent, non-adhesive particles such as inert glass beads or the same resin are used together with a hydrophilic colloid adhesive such as gelatin or polyvinyl alcohol to form a porous layered structure. However, this is because the porosity of the porous layer changes depending on the amount of the adhesive, and if hydrophilic colloid is added to an extent that contains sufficient adhesive strength, the porosity decreases and the porosity of the fluid sample decreases. It obstructs the flow, and conversely, if it is reduced, the layered structure becomes so fragile that it cannot be pressed. Furthermore, hydrophilic colloids serving as adhesives have the disadvantage that, because they are water-soluble, they cause a further reduction in adhesive strength in the presence of aqueous fluid samples. -! Moreover, the porous layer disclosed in the same patent has the disadvantage that many large complex macromolecules and cells contained in the fluid sample tend to clog in the pores and completely obstruct the flow of the fluid. are doing.

Also, U.S. Patent Nos. 2,297,247 and 2.7
45,141 discloses a layer of agglomerated particles that is thermally softened or solvent softened and hardened. In this layer, the particles fuse together at points of mutual contact. This means that all the particles constituting the aggregated particle layer disclosed in the above patent are susceptible to deformation due to thermal softening or solvent softening, and have the disadvantage that the desired interparticle voids are reduced or completely eliminated. It shows. US Patent No. 2
, 297,248 discloses a filter element which is a particulate structure bonded with a particulate layer "appropriate cement".

However, this also tends to fill the interparticle voids depending on the amount of adhesive, which is easily clogged with large complex macromolecules and cells, and easily impedes the flow of the fluid. It also has the disadvantage that the flow is delayed. Furthermore, JP-A-55-90859 discloses a porous cohesive three-dimensional lattice in which non-swellable, liquid-impermeable, heat-stable organic polymer particles are bonded together using a polymer of a different type from the polymer particles as an adhesive. A particulate structure is disclosed.

Similar to the above-mentioned patents, the above patent also uses an adhesive polymer with low thermal stability, that is, a low glass transition temperature (hereinafter abbreviated as Tg), which is thermally softened at a temperature higher than k Tg to bond the thermally stable organic polymers together and create an interconnecting space. The entire granular structure is formed. Therefore, if the amount of adhesive used to completely form the granular structure described in the above patent publication is large, the porosity will be reduced, and if the amount is too small, sufficient adhesive strength will not be obtained. Due to the amount of the above adhesive used, all of it must be placed in the desired position between all of the above heat-stable polymer particles, and it is difficult to control a constant porosity throughout. In addition, it has the disadvantage that the adhesive strength is low because the inert beads are only adhesively bonded by deformation due to thermal softening of the adhesive.

Therefore, an object of the present invention is to provide an analytical element that has a spreading layer made of a particle bond formed from a particle structure with high adhesive strength, which makes it easy to completely control a constant porosity, and has excellent quantitative performance. Our goal is to provide the following.

As a result of intensive studies, the inventor Hiromu was able to overcome the above-mentioned drawbacks by using an analytical element consonant having the following structure.

That is, to summarize the present invention, the present invention provides an analytical element for analyzing all components in a fluid sample, which includes at least one fibrous structure layer and at least one particle binder layer having a reactive group. The particle aggregate layer is composed of a particle aggregate that is a non-swellable three-dimensional lattice having interconnecting voids with a porosity of 25 to 85% to allow complete transport of the fluid, and the particles are non-swellable to the fluid. The fibrous structure layer is a heat-stable organic polymer particle having a size of 1 to 550 μm that is swellable, impermeable, and has a reactive group, and the fibrous structure layer is coated with a fiber dispersion. The present invention relates to an analytical element characterized in that it is formed by machining.

Hereinafter, the analytical element of the present invention will be explained in more detail.

Each of the above layers according to the present invention is used to contain reagents that cause a quantitative reaction with a sample component to be analyzed, and to perform the entire quantitative reaction within the layer.

The particle conjugate layer in the present invention is composed of a particle conjugate which is a non-swellable three-dimensional lattice having interconnected voids with a porosity of 25 to 85 cm to enable the total transport of the fluid sample, and the particles are These are heat-stable organic polymer particles having a size of 1 to 550 μm that are non-swellable and impermeable to fluids and have a reactive group, and in the present invention, these polymer particles are Is it directly chemically bonded to the above reactive group in the contact area, or is it a low molecular compound due to the above reactive group in the bonding area between particle units? It is preferable that the chemical combination be made through (9).

The particle conjugate layer of the present invention has a porous structure that binds many high molecular weight substances dissolved or dispersed in a fluid sample, particularly a biological fluid sample, blood cells of red blood cells, or interactive compositions used in fluid analysis procedures. They can be easily accommodated or separated without creating blockages or substantially interfering with fluid transport.

The analytical element of the present invention is an object to be analyzed (hereinafter referred to as an analyte).
It can perform a very effective diffusion function for liquids containing either low molecular weight or high molecular weight substances. That is, these devices can easily accommodate the applied fluid sample across a variety of analytes, can be uniformly distributed within the analytical device, can be weighed, and can be easily transported using particle conjugates. Has a structure.

The particle bond layer consisting of a particle bond formed from particles of a thermostable organic polymer particle unit having a reactive group of the present invention is formed by the reaction between the particle units at the contact portion of the particle units as described above. (10) The reactive groups react with each other to form a three-dimensional lattice, or the reactive groups of the particle units react with each other via a low-molecular compound at the bonding part of the particle units. A three-dimensional lattice is generated, and the particle units are bonded to each other by strong chemical bonds. Therefore, the strength of the layer made of the particle bond is sufficient to withstand external physical forces, It is clear that this is sufficient to maintain the entire structure.

In the present invention, the organic polymer particle units preferably have a size of about 1 to 550 microns, and these particle units form a particle assembly that is a three-dimensional lattice containing all interconnected voids, and The fluid-impermeable, non-swellable particles of the present invention having a total void volume of 25 to 85 inches indicate that the fluid is substantially impermeable, and non-swellable means that the fluid does not substantially permeate when in contact with the fluid. 0 refers to those that do not exhibit swelling properties.
The degree of swelling is, for example, A, G-y (A-G
reen) and G9 Engineering, P.

(1) Journal of Photographic Science (G,■, P+Levenson)
Journal of Photography, c
5science), Vol. 20, p. 205 (1972), can be used to measure under the desired fluid.

That is, on a suitable support such as a polyethylene terephthalate support, (1) a self-supporting film of an organic polymer under consideration for use as a particle material;
The entire layer with a dry film thickness within the range of 50μ is formed, and the film or layer is immersed in a liquid bath with a weight of 58°C for about 25 minutes using the swelling needle, and the increase in the thickness of the film or layer is measured. . The degree of swelling measured by these methods is approximately 2
Less than 0%, preferably less than about 10% purple, can be used as the preferred organic polymer particle material. Although it is possible to vary widely within the range (12y) and it is also possible to use a mixture of various sizes, in a preferred embodiment, these particle units have a substantially uniform size.

Preferably, the particle unit surface is curved, more preferably substantially spherical. The size of the voids contained in the layer is regulated to some extent by the size of the organic polymer particle unit. In a preferred embodiment for applying fluid samples containing completely cellular structures such as red blood cells in the range of 6 to 8 microns, relatively large particle units are used.
In such a case, 20 to 500 μ, preferably 20 to 15
It is possible to use one with a size of 0μ.

1 to 100 for the transport of large complex molecules such as macromolecules of biological origin, such as riboproteins, antigens or antibodies.
μ, preferably 2 to 50 μ, more preferably 2 to 20 μ
The range is on the order of magnitude. Even smaller molecular analytes, such as glucose molecules, uric acid molecules, etc.? For aqueous fluids containing 15 to 50 μm in size.
) can exist together.

A direct chemical bond between adjacent particle units, which is one of the preferred embodiments of the present invention, may be formed by a reaction between adjacent particle units having the same type of reactive group, for example, particle units having epoxy groups. However, it is also possible to form the reaction between particle units having different types of reactive groups, for example, a particle unit having an epoxy group and a particle unit having an amine group. Molecular compound? The chemical bond between adjacent particle units via a diamino compound may be formed by a reaction between an adjacent particle unit having the same type of reactive group and a low-molecular compound, such as a particle unit having an epoxy group, and a diamino compound. It is also possible to form the particles by the reaction of a particle unit having different types of reactive groups and a low-molecular compound, for example, a particle unit having separate amino groups and carboxyl groups, and a bisformyl compound. Is the particle unit two or more reactive groups? 1J1 may be included. In order to chemically bond all the reactive groups directly or with a low-molecular compound, heating may be performed as necessary, or catalytic sound may be used. Particle units having reactive groups can be obtained, for example, by homopolymerizing or copolymerizing monomers having reactive groups or their precursors.

Although it has been described above that each particle unit may have two or more types of reactive groups, particle units having two or more types of reactive groups may have, for example, different types of reactive groups or precursors thereof. It is possible to carry out total copolymerization of the monomers. When a monomer having a precursor of a reactive group is used, for example, after forming a particle unit, the particle unit having the reactive group can be formed by, for example, hydrolysis.

In the present invention, the monomer unit having a reactive group preferably has a polymer particle unit width of 1 to 50% by weight, particularly preferably 0.5 to 20% by weight.

More detailed explanation regarding the direct chemical bond between adjacent particle units, which is one of the preferred embodiments of the present invention (15) Then, - Direct chemical bond is formed by reaction between adjacent particle units having the same type of reactivity as described above. Examples of monomers having a reactive group suitable for this include monomers having an epoxy group, monomers having an aziridyl group, monomers having a formyl group, monomers having a hydroxymethyl group, and monomers having an isocyanate group. monomers having a thiol group, monomers having a carbamoyl group, and monomers having a carbamoyl group.

Examples of monomers having all epoxy groups include glycidyl acrylate, glycidyl methacrylate, allyl glycidyl ether, and 4-vinylcyclohexane monoepoxide. Examples of monomers having an aziridyl group include aziridylethyl methacrylate, 1-ethylenesulfonylaziridine, 1-ethylenecarbonylaziridine, and aziridylethyl acrylate. Examples of the formyl group-containing monomer include acrolein and methacrolein. Examples of monomers having hydroxymethyl foundation (16) include N-methylol acrylamide, N-methylol methacrylamide, N-methylol diacetone acrylamide, and the like. Examples of the monomer having an incyanato group include vinyl isocyanate, allyl isocyanate, and the like. Examples of the monomer having a thiol group include vinylthiol, p-thiolstyrene, m-thiolstyrene, vinylbenzylthiol, and acetyl forms thereof. Examples of monomers containing carbamoyl groups include acrylamide, methacrylamide, maleamide,
Examples include diacetone acrylamide.

Other monomers to be copolymerized with the monomer having a reactive group can be arbitrarily selected as long as the resulting particles satisfy the conditions of liquid impermeability and non-swellability.

Examples of combinations of different types of reactive groups used when forming a chemical bond by reaction between particle units having different types of reactive groups described above include (17,1 D, H, solomoy (D, H.Solomon)
Author [The Chemistry of Organic Film Formers J ("The Oh, emist")
ry of Organic Film Formere
-3 (1967). Specifically, for example, an epoxy group and an amino group, a carboxyl group and an amino group, a carbamoyl group and a hydroxymethyl group, a carbamo-yl group and a tomethoxy group, a hydroxymethyl group and a carboxymethoxymethyl group, a hydroxyl group and a carboxyl group, and an epoxy group and 100004H, (t) group, ureido group and carboxyl group, formyl group and hydroxyl group,
Examples thereof include a vinylsulfonyl group and an amino group, a haloethylsulfonyl group and an amine group, an active methylene-containing group and a formyl group, an epoxy group and a carboxyl group, an aziridyl group and an amino group, and an aziridyl group and a carboxyl group.

Among the monomers having the various reactive groups mentioned above, specific examples of the monomers having an epoxy group, an aziridyl group, a hydroxymethyl group, or a carbamoyl group include those mentioned above. Other reactions (18) Reactive groups? Examples of monomers having all 0 carboxyl groups shown below include acrylic acid, methacrylic acid, iconic acid, maleic acid, itaconic acid bovine ester, maleic acid half ester, etc. Examples of the monomers present include aminostyrene, N, N-
Examples include dimethylamine ethyl acrylate, N,N-dimethylamine ethyl methacrylate, and the like.

Examples of the monomer having a methoxy group include methoxyethyl acrylate, ethoxyethyl acrylate, methoxyethyl methacrylate, and ethoxyethyl methacrylate.

Monomers containing -Coo('1i4H, (t) i include, for example, t-butyl acrylate, t-butyl methacrylate, etc. Monomers containing 0 ureido groups include, for example, ureidoethyl acrylate. , ureidoethyl methacrylate, ureido vinyl ether (e.g., formula OH
,=0HONRC! Examples include those represented by 0NHR', where R is a hydrogen atom or a methyl group, and R' is a hydrogen atom or a lower group such as methyl or ethyl (representing a 19 parkyl group). Examples of monomers having a hydroxyl group include 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate,
Examples include 2-hydroxyethyl acrylate and 2-hydroxypropyl acrylate. ! - Examples of the monomer having a loethylsulfonyl group include chloroethylsulfonylethyl methacrylate, furomoethylsulfonylethyl methacrylate, and the like. Examples of monomers having a vinylsulfonyl group include 2-(vinylsulfonylamino)ethyl methacrylate and vinylsulfonylmethylstyrene. Examples of monomers having a zero-active methylene group include acryloyl acetone, methacryloyl acetone, etc. Can be mentioned. Examples of monomers having a carboxymethoxymethyl group include N-carboxymethoxymethyl-acrylamide, N-
Examples include carboxymethoxymethyl-methacrylamide.

(2U) When used on a particle unit having two or more types of reactive groups as described above, a chemical bond due to the above-mentioned same type of reactive groups and a chemical bond due to different types of reactive groups occur. It is also possible to produce a particle bond by using two or more types of particle units having reactive groups.Next, in another preferred embodiment of the present invention, the chemical bonds between adjacent particle units are To explain in more detail what is carried out in compound Kanakura, the above-mentioned direct chemical reaction bond is used as a monomer having a reactive fund suitable for chemical bonding between adjacent particle units to be carried out in the entire low molecular weight compound. Examples of monomers having a reactive group suitable for forming a total monomer include all the monomers mentioned above. It is possible to inspire. For example, gelatin hardeners commonly used in the photographic field can be used as the above-mentioned low molecular weight compound.

(21) Furthermore, examples of low-molecular compounds that chemically bond with monomers lacking epoxy groups include bisphenol compounds (bisphenol A, etc.), dicarboxylic acid compounds (coccic acid, etc.)
, amino compounds (methanediamine, diethylenetriamine, ethylenediamine, n-hexylamine, etc.).

Monomers having an aziridyl group can also be used in the same manner as the above-mentioned low molecular weight compounds. Examples of low-molecular compounds that chemically bond with monomers having a carboxyl group include bisepoxy compounds (for example, hexamethylenebisoxirane, etc.), epihalohydrin compounds (epichlorohydrin, etc.), glycolated compounds (ethylene glycol, etc.), and dihydroxy compounds 70 to 400. Dinitrile compound, (22) 1. and polyvalent metal salts, such as A1. , Ba-, Oa
, Sr, Mg, and pb can also be usefully used.

A dialdehyde compound (eg, glutaraldehyde, etc.) can be advantageously used as a low molecular compound that chemically bonds with a monomer containing an active methylene-containing group.

Examples of low-molecular compounds that chemically bond with monomers having amino groups include aldehyde and dialdehyde compounds (
mucochloric acid, glutaraldehyde, etc.), diisocyanate compounds (hexamethylene diisocyanate, etc.), bisepoxy compounds (hexamethylene bisoxirane, etc.)
It is possible to use disulfonyl chloride compounds (such as phenol-2,4-disulfonyl chloride).

The monomer having a reactive group of the present invention and the low-molecular compound chemically bonded to the monomer may be appropriately selected from a wide range of combinations depending on the reactivity of each monomer and other purposes. (25) For example, H, Nromoy (D, Hosolomo
n) Author [The Chemistry of Organic Phil A-7 Omars J (-The Chemis
try ofOrganic Fi], m Forme
rs”) Jo7-Wiley & Sons Co., Ltd.
(1967) can also be used advantageously, but the combination of the above-mentioned monomer having a reactive group of the present invention and a low-molecular compound chemically bonded to the monomer is an example of a preferred embodiment. However, this does not limit the present invention in any way.

The low molecular weight compound of the present invention is about 2 times to 100 times the amount of the monomer having a reactive group in the polymer particle unit of the present invention.
It is possible to use 5 times the molar amount, but preferably about 1.
It is 5 times molar to α01 times molar.

Examples of other preferable monomers to be copolymerized with the monomer having the above-mentioned reactive group are shown below.

(1) (24) [In the formula, R1 and RZ may be the same or different, and do not contain a hydrogen atom, a rogen atom, or a substituted or unsubstituted amino group containing 1 to 10 carbon atoms. Full representation of non-hazardous substituents such as alkyl groups or aryl groups R
3 represents a hydrogen atom, a halogen atom, a substituted or unsubstituted aliphatic group having 1 to 10 carbon atoms, or an aromatic group not containing an amino group. ] Examples of aliphatic groups and aromatic groups include alkyl groups, alkoxy groups, aryl groups, and aryloxy groups. Examples of the monomer represented by formula (1) include styrene, vinyltoluene, vinylbenzylchloroIJ)'', and t-7'tylstyrene.

(I) OHR6= OR4-00OR11 [In the formula, R6 is the same as R1 in formula (1), R4 is a hydrogen atom or a methyl group, and H& is a substituted or unsubstituted aryl group or alkyl group each having 1 to 10 carbon atoms. groups, alkalis (two monol groups and aralkyl groups) (1) Polymerizable unsaturated nitrile monomers such as acrylonitrile and methacrylonitrile.

(M Particulate crosslinkable monomers with two addition polymerizable groups, such as divinylbenzene, N,N-methylenebis(acrylamide), ethylene diacrylate and hyethylene dimethacrylate.

By appropriately combining and copolymerizing these monomers and the monomer having the above-mentioned reactive group, it is possible to constitute the polymer particle unit of the present invention. The particle units are preferably Ω to 99,5% by weight for these monomer units 't(11, (1) and (1), respectively, and 0 to 10% by weight for (IV)). is preferably contained in an amount of 0 to 5% by weight.

Typical specific examples of the polymer particle units forming particle combinations of the present invention are shown below, but the present invention is not limited thereto. Also, [] after each exemplified compound
(26) 0 indicates the weight percent of the monomer used in the polymerization reaction (Exemplary Compounds) (1) Styrene-glycidyl methacrylate copolymer (90/10).

(2) Styrene-methyl acrylate-glycidyl methacrylate copolymer (80/1515).

(3) Styrene-n-butyl methacrylate-glycidyl methacrylate copolymer [75/15/10].

(41 Styrene-p-vinylbenzyl chloride-glycidyl methacrylate copolymer [80/10/1 (
+).

(5) Styrene-mixed divinylbenzene-glycidyl acrylate copolymer (90/2/8).

(6) p-vinyltoluene-glycidyl methacrylate copolymer (90/10).

(7) Methyl methacrylate-glycidyl methacrylate copolymer (80/20').

(8) Styrene-N,N-dimethylaminoethyl methacrylate copolymer (q 5./s').

(27) (9) Styrene-aziridylethyl methacrylate copolymer (9s15').

00 Styrene-methyl acrylate-acrolein copolymer (901515).

On Styrene-acrylamide copolymer [9515
].

(2) Styrene-vinylthiol copolymer (9515
].

(To) Styrene-methylolated acrylamide copolymer (9515).

α◆ Styrene-t-butyl acrylate-glycidyl methacrylate copolymer [951515].

(g) Styrene-vinyl isocyanate copolymer (?
515)0 (2) Methyl acrylate-styrene-N-methylolacrylamide copolymer (50155/15).

(b) Styrene-glycidyl methacrylate-N, N
-dimethylaminoethyl methacrylate copolymer (:
901515').

(28) 6 barrels Styrene-methacrylic acid-acrylamide copolymer (95/215).

O1 styrene-N-methylolacrylamide-methoxyethyl acrylate copolymer [901515].

Hp-vinyltoluene-N-methylolacrylamide-acrylic acid copolymer (9078/2).

Ql) Methyl methacrylate-glycidyl methacrylate-1-butyl acrylate copolymer [80/10
/10].

) Styrene-p-vinylbenzyl chloride-acrylic acid-ureidoethyl acrylate copolymer (75/1
0/5/10).

(c) Styrene-methacrolein-1-hydroxyethyl meccrylate copolymer (901515).

041 Styrene-acrolein-acetoacetoxyethyl methacrylate copolymer (8515/10) 0 (29) (c) Styrene-N,N-dimethylaminoethyl acrylate-vinylsulfonylethyl methacrylate copolymer (901515).

(E) p-vinyltoluene-aminostyrene-vinylsulfonylethyl methacrylate copolymer (85/1
0/5'J.

(5) Styrene-N,N-dimethylaminoethyl methacrylate copolymer (90/10).

(To) Styrene-acrylic acid copolymer (9715).

Kan styrene-acrylamide copolymer [9715].

OI p-vinyltoluene-t-butyl acrylate copolymer (9515).

cll) Methyl acrylate-methacrylamide copolymer (95/5).

(2) Styrene-N-methylolacrylamide copolymer (95/5).

01 p-vinylbenzyl chloride-N-methylolacrylamide copolymer (96/4).

(Foundation) Styrene-Itakoshi polymer (98/2).

(50) C151 styrene-t-butyl acrylate copolymer 1
”92/8).

(To) Methyl acrylate-styrene-acrolein copolymer [50/6515].

Oη Methyl methacrylate-styrene-2-hydroxyethyl methacrylate copolymer (25/70/
5) 0 (to) Styrene-vinylsulfonylethyl acrylate copolymer [80/20].

Ol Styrene-N,N-dimethylamine ethyl acrylate copolymer (90/101°) Styrene-methyl acrylate-acetoacetoxyethyl acrylate copolymer (901515).

(41) Styrene-methacrylic acid copolymer (951
5) 00% styrene-N,N-diethylaminomethyl acrylate copolymer (97,5/2.5).

(41 styrene-glycidyl methacrylate-N, N-
Dimethylaminoethyl methacrylate copolymer (90/
5/5].

(51) Among the above-mentioned exemplified compounds, examples of those having one type of reactive group (exemplary compounds (1) to 0→ monomer units each having a different reactive group? Two or more types of identical particle units) Examples of what is included in the above are the compounds (b) to (c), and -! In addition, the present invention 1d: an organic polymer particle unit containing a monomer unit having a kind of reactive group; Organic polymer particle units containing a monomer unit having a type of reactive group different from the above-mentioned reactive group can be used in isophones, and typical examples of combinations are as follows. A) Exemplary compound (1) and Exemplified compound B) Exemplified compound (C) and Exemplified compound (2) C) Exemplified compound (1) and Exemplified compound (G) D) Exemplified compound I3D and Exemplified compound 02E) Exemplified Compounds (G) and Exemplified Compounds (G)
F) Exemplified Compound (1) and Exemplified Compound (C) G) Exemplified Compound (G) and Exemplified Compound C17) H) Exemplified Compound (To) and Exemplified Compound 0 Exemplified Compound (40 and Exemplified Compound α0)
(52) J) Exemplary Compound G11) and Exemplary Compound (1) Synthesis examples of exemplary compounds of the present invention are shown below, but the present invention is not limited thereto.

Synthesis Example-1 Synthesis of Exemplified Compound (1) Styrene 90 parts, glycidyl methacrylate 10 parts, 2
,2'-azobis(2,4-dimethylvaleronitrile)
A mixture of 5 parts of monomer and a polymerization initiator; 5 parts by weight of tricalcium phosphate, 0.04% by weight relative to the above monomers;
An aqueous solution of sodium dodecylbenzenesulfonate in 700 d of IC, TK-Homojitsu, Tar (manufactured by Tokushu Kika Kogyo Co., Ltd.) was added while stirring at a stirring speed of 5000 rpm. After addition, stir for about 50 minutes,
While observing with a microscope, when the particle size reached about 20μ,
Mixture in a 4-head flask with a regular stirrer (Ikari type), cooling tube, nitrogen gas introduction tube, and thermometer? Put in, 2
The stirring speed was changed to 00 rpm, and the entire polymerization reaction was carried out at 60°C for 8 hours under a nitrogen gas stream until the reaction was completely completed.
(55) Next, the entire contents were cooled to room temperature, and tricalcium phosphate was decomposed and removed with dilute aqueous hydrochloric acid solution.1. The polymer particles wP were separated by repeated washing with water and dried to obtain polymer particle units having an average particle size of 18 μm.

Synthesis Example 2 Synthesis of Exemplified Compound (3) 75 parts of styrene, 15 parts of n-butyl methacrylate, 10 parts of glycidyl methacrylate and 2.2-7 zobis(
A mixture of 5 parts of monomer (2,4-dimethylvaleronitrile) and a polymerization initiator was added to a mixture consisting of 2% by weight of tricalcium phosphate and 2% by weight of sodium dodecylbenzenesulfonate based on the monomers. The solution was added to 700 ml of the aqueous solution while stirring using a TK-Homojetter sound at a stirring speed of 2 DOO rpm. After addition, 50
Stir at the same stirring speed for 1 minute, observe with a microscope, and when the droplets of the monomer mixture have a particle size of about 100μ,
The same reaction and operation as in Synthesis Example-1 were carried out, and the average particle size was 10.
A polymer particle unit of 0μ was obtained.

(54) Synthesis Example-5 Synthesis of Illustrated Compound @Syrene 97.5 parts of styrene, 2.5 parts of N,N-diethylaminomethyl acrylate and 2,2'-azobis(2゜4-
5 parts of dimethylvaleronitrile) were dissolved to prepare a monomer mixture. Next, a water-soluble 700 consisting of 5 monomers of hydrophilic silica Aerosil 200 (manufactured by Tegusa) was prepared for the above monomer, and the above aqueous solution was processed into a TK-homogenerator and stirred at a stirring speed of 600 Orpm. While stirring, the above monomer mixture was added. After addition, about 50
Stir for 1 minute at the same stirring speed, and when it is dispersed into droplets of approximately 207 tons for microscopic observation. Pour the dispersion into a flask and stir at 60°C under a nitrogen gas flow at a stirring speed of 250 rpm.
After the polymerization reaction was carried out for an hour and the reaction was completed, the contents were cooled to room temperature and diluted with carbonic acid. Polymer particle units with average particle diameters of 1 and 5 microns were obtained.

Synthesis Example 4 Synthesis of Exemplified Compound 5 parts of 2,2-azobis(2,4-dimethylvaleronitrile) was dissolved in 95 parts of styrene and 5 parts of methacrylic acid to prepare a monomer mixture. Then 3 weight -
An aqueous solution of 700 di of aluminum oxide (manufactured by Tegusa) was prepared and processed in a TK-homogenerator.
The aqueous solution was added over the monomer mixture while stirring at a stirring speed of 0 rpm. After the addition, stirring was carried out for about 50 minutes at the same stirring speed, and when the particle size of the monomer mixture droplets became about 15μ by microscopic observation, the same reaction and operation as in Synthesis Example 5 were carried out, and the average particle size was One polymer particle unit with a diameter of 16 μm was obtained.

The above thermostable organic polymer particle unit containing a reactive group according to the present invention typically has a Tg of 50°C or higher, preferably a Tg of 40°C or higher (56). Tg as used herein means the temperature at which a polymer changes its state from a glass state to a rubber state or a fluid polymer, and is an index of thermal stability. For example, Techniques and Methods of Polymer Evaluation
es and Methods of Po'lymer
Bvaluation) Volume 1, Marcel Dekker,
It can be measured according to the method described in N. Y. (1966).

The thickness of the fiber structure layer according to the present invention can be arbitrarily selected depending on the purpose, but is preferably about 50 μm to about 6 μm.
00μ, more preferably about 50μ to about 400μ.

The fiber structure layer according to the present invention described above is
Needless to say, it does not have a mesh or woven structure as described in JP-A-6551 or JP-A-55-164556, and the free pore area is substantially zero.

The material (57) for forming the fiber structure layer according to the present invention includes natural cellulose or its derivatives, and synthetic fibers such as polyethylene, polypropylene, and boria. In addition, as the material for forming the fiber structure layer, one or more materials of any size can be selected. 50 mesh ~ 525 with 8 standard sieve
The mesh size is preferably 100 to 520 meshes, more preferably 200 to 500 meshes.

To produce the particle combination layer and the fiber structure layer in the analytical element of the present invention, the layer-forming material is dispersed in a total dispersion medium.The dispersion medium used includes a liquid carrier, a surfactant and It is preferable to use one in which the entire organic polymer is added.

The liquid carrier may be an aqueous liquid.

However, the layer forming material, which is a particle unit or a fiber,
Other liquid carriers can also be used, such as various organic liquids, provided that they are insoluble in the liquid carrier (58) and therefore their structural properties are preserved.

Typical liquid carriers other than water plates include water-miscible organic solvents, aqueous mixtures of water and water-miscible organic solvents, and suitable water-immiscible organic solvents. Water-miscible organic solvents include lower alcohols (ie, alcohols with an argyl group of 1 to 4 carbon atoms), acetone, and tetrahydrofuran.

Water-immiscible solvents include lower alkyl esters, such as ethyl acetate, and halogenated organic solvents, such as halogenated hydrocarbons (eg, chloroform, methylene chloride, carbon tetrachloride, and the like).

As usable surfactants, it is possible to use both ionic (anionic or cationic) and nonionic surfactants, but preferably nonionic surfactants are effective. It is. Examples of nonionic surfactants include alkyl-substituted phenol polyethylene glycols such as 2,5-di-t-butylphenoxypolyethylene glycol, p-octylphenoxypoly(59glycidyl ether, and p-isononylphenoxypolyethylene glycol). Examples include alkylene glycol surfactants, polyalkylene glycol esters of higher fatty acids, etc. These surfactants have the effect of regulating the permeation rate of the fluid sample and at the same time suppressing the occurrence of undesirable "chromatographic phenomena." Furthermore, the surfactant has the effect of reducing the undesirable effects of various proteins contained in the biological fluid sample.

The surfactants can be used in widely selected amounts, but may be 7 to 10 times the weight of the particle unit or fiber.
Weight f#ch~flo05wt%, preferably 6wt%~0
．． The organic high molecular weight polymer is not only useful as a dispersion stabilizer for the dispersion, but also as a binder when forming the twill fiber structure layer of the present invention. However, it is preferable to use all kinds of natural or synthetic polymers.

(UO) Preferred organic polymers that can be used include, for example, the organic polymers shown as particle units forming the entire particle combination layer, gelatin, gelatin derivatives such as modified gelatin, polyvinyl alcohol, polyvinyl Examples include pyrrolidone, polycarbonate, polyacrylamide, cellulose acetate, and polyvinyl butyral.

The above-mentioned organic high molecular weight polymer can be used in an amount selected from a wide range, but an amount that does not fill a substantial part of the interstitial volume formed by the three-dimensional entanglement of fibers is preferable, and the amount of the organic polymer can be selected from a wide range. 10% by weight based on fiber weight ~ α0
05% by weight\suitably 6% by weight - [L05% by weight can be used.

When applying the organic polymer to the dispersion medium for forming each layer in the analytical element of the present invention, various methods can be used. For example, the organic polymer can be used by dissolving it in the liquid carrier, or it can be used by dispersing it in a liquid carrier in which the organic polymer does not completely dissolve (for example, finely powdered (41) latex). preferable.

As previously discussed, each layer is in fluid contact with each other. In this specification, the expression "fluid contact" is used to refer to layers that cooperate with each other in such a manner that, under conditions of use, fluid (liquid or gaseous) can pass from one layer to another. . Preferably, such fluid contact performance is uniform along the contact interface between the fluid contact layers. The fluid contacting layers may be contiguous or separated by intervening areas. However, such intervening areas are also in fluid contact and do not impede fluid passage. In some cases it may be desirable to use areas that are initially spaced apart within the device. In such cases, the analytical element of the present invention can be supported on a support as described above, with fluid contact of the layers being effected substantially at the time of sample application, for example by full compression of the element.

Useful support materials such as vinegar ff cellulose, polyethylene terephthalate, polycarbonate and polyvinyl compounds (eg polystyrene) (42
) Materials include glass, metal, and paper. The preferred support for a given device is one that is compatible with the mode of result detection.

For example, in fluorometric detection where fluorescent emissions within the device are transmitted from within the device through the support to an external detector, it is desirable to use a material that exhibits only a low degree of background fluorometric emission as the overall support material.

The analytical element of the present invention may be provided with layers conventionally used in combination with these analytical elements, such as a reagent layer, a reflective or radiation blocking layer, a registration layer, a scintillation layer, and a cleaning layer. be.

If any of the layers, such as a reagent layer, a reflection or radiation blocking layer, and a registration layer, are present in the device, these layers are usually interposed in the device between the support and each layer of the invention. is preferred.

Further, the radiation blocking agent may be one of various known compounds, and may be contained within the organic polymer particle unit in the analytical element (45) of the present invention. The amount is 0.5 to 0.5 to 0.5 to
It is possible to set it as 60 weight%.

In manufacturing the analytical element of the invention, the individual layers are preformed (
~, then laminating them prior to use, or
It is also possible to hold the element as a separate member until it is in fluid contact during use. If the layer preformed as a separate component can be coated, it can advantageously be assisted from a solution or dispersion. The surface of the layer is the surface from which the layer can be physically peeled off when dry. However, if adjacent layers are desired, a simple method which avoids the problem of having to carry out multiple peeling and lamination steps 7 is to peel off the entire first layer, coat it on the surface or on the support and, if desired, The analytical elements of the present invention can then be coated directly on or next to the precoating layer by a subsequent layer. 2681
It can be coated by various coating methods such as extrusion coating using a hopper as described in U.S. Pat.
No. 791 and British Patent No. 857,095. The analytical element of the present invention can be applied not only to the field of clinical chemistry but also to other fields of chemical analysis. It is possible and
The ability to retain a constant fluid within a constant membrane area can also be used in combination with other functional layers (eg, layers of photographic elements).

The analytical element of the present invention is extremely advantageous for clinical chemical analysis of body fluids such as blood, serum, lymph, and urine. Particularly in the case of blood analysis, serum is usually used, but the analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage.

If whole blood is used, a radiation blocking layer or other reflective layer can be used if desired to prevent interference of the radiation for detection (45) by blood cells. In the case of directly observing the color of blood cells, for example in the case of hemoglobin analysis, it is of course unnecessary to remove the gold reflective layer.

After manufacturing the analytical element of the present invention and obtaining analytical results as detectable changes, reflection spectrometry, transmission spectrophotometry, emission spectrophotometry, or fluorescence spectrophotometry can be performed in response to various types of detectable changes. , or measured by scintillation measurement, etc. By applying the measured values obtained in this way to a calibration curve prepared in advance, the total amount of unknown apple substances can be determined.0 Immunological analysis is sufficient for qualitative or quantitative analysis of antibodies and antigens. This is a method recognized by

All immunoassays are based on the unique (46) immunological phenomenon in which % different antibodies recognize and bind to specific antigens. Examples of these include radioimmunoassays, enzyme Examples include immunoassay method and fluorescence immunoassay method.

The preparation methods, measurement methods, and measurement principles of the antibodies, antigens, and labeled antigens or labeled antibodies used in these measurement methods are detailed in the book, but for example, the "Irie Solid Line 1 Radioimmunoassay" Kodansha (1974) ), Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai (eds.) “Enzyme Immunoassay” Igaku Shoin (19
1978).

In the immunoassay method commonly used to perform the above measurements,
It has various drawbacks.

However, many drawbacks are overcome by applying the immunoassay to the analytical element of the present invention. In addition to the above-mentioned immunoassay using the basic principle of antigen-antibody reaction, the present invention also includes an immunoassay based on antigen-antibody displacement interaction as described in West German Patent Application Publication No. 280145. (47) This is self-evident.

Additionally, an amount of antibody directed against the labeled antigen is incorporated into the analytical element and this antibody is immobilized. Preferably, this immobilization takes place within the layer of the device with the particle conjugate layer. Immobilization can be achieved by adsorbing or chemically bonding the antibody to the surface of the organic polymer particle unit of the particle conjugate layer. The fluid sample to be analyzed for unknown antigen is then contacted with the device in the presence of whole labeled antigen. Labeled antigens can be associated with immunoassays in one of several ways, including, among others:

That is, 1) add labeled antigen '5r directly to the fluid sample (containing unlabeled antigen), then apply the fluid sample containing the labeled antigen to the immunoassay element for analysis; 2) add the label to the immunoassay element; adding antigen and fluid sample separately (e.g. (a) adding labeled antigen just before or after adding fluid sample; and (b) adding labeled antigen' to the element;
followed by drying and rewetting the device upon addition of the fluid sample); (4B); label antigen whole immunoassay device so that the fluid sample can be started by simple application; For example, a labeled antigen can be incorporated into a single reagent layer of a device or a single reagent layer of a device containing an immobilized antibody. In any case, when incorporating a labeled antigen into an entire device, care must be taken to keep the labeled antigen separate from the immobilized antibody and to avoid any premature binding of the labeled antigen to the antibody.

When a fluid, sample, in the presence of co-labeled antigens as described above is contacted with the whole immunoassay device, the labeled and unlabeled antigens (which are present in the sample and are the unknowns to be measured) are absorbed into the layers of the device. competitively binds to antibodies that are immobilized within the body. Useful measurement methods that can be used to determine the presence and/or concentration of unlabeled antigen include the following. (m) Detection of unbound labeled antigen migrated to a second layer of the device, such as a registration layer.

or (B) detection of bound labeled antibody bound to immobilized antibody. In either case, the culprit (or analyte) of the unlabeled antigen (49) in the fluid sample can be determined based on the concentration of labeled antigen detected.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited thereto.

Example 1 A layer of deionized gelatin with a dry film thickness of about 20 μm was coated on a transparent subbed polyethylene terephthalate support with a film thickness of 180 μm, and the particles of the present invention with the composition shown in Table 1 were coated on the upper layer. A binder layer and a comparative porous spreading layer were provided for each of the resistors 1, It, LIV and comparative elements 1. II was formed.

Table 1 (51) The following test session was conducted on the device according to the present invention and the comparative device shown in Table 1 above.

That is, a cellophane tape having a length of 5α was pasted on the particle combination layer and porous spreading layer of the above element and comparative element, and after one end of the tape was peeled off, a peeling strength test was conducted. The evaluation is based on the degree of peeling: 0Items that did not peel off at all...AO cellophane tape? If less than half of the bulge was peeled off...B (52) If the entire part where the O cellophane tape was peeled off was peeled off...C If more than the part where the O cellophane tape was peeled off, it was rated D.

Similarly, a red dye (Br11.1
ant 5carlet 5R) fl 05% by weight
10μ of the added and colored fluid sample was dropped, and the accommodation time of the fluid sample in the layer and the change in the shape of the layer were observed. The results are shown in Table-2.

Table 2 (55) As shown in the results shown in Table 2, comparative elements I and ■
Both have weak mechanical strength, and especially in the case of comparative element H, the structure 1 of the porous developed layer cannot be maintained by applying a fluid sample. It can be seen that the transport function cannot be fully fulfilled.

On the other hand, elements I to IV having the particle combination layer of the present invention are
It can be seen that it has an interconnecting cavity for transporting the fluid sample, and its mechanical strength is excellent, and the time to accommodate the fluid sample in the layer is extremely fast.

Example 2 On a polystyrene support that exhibits only low levels of fluorescence,
Each layer consisting of the following composition was sequentially coated onto the above support, first, 216 mW of deionized gelatin was applied.
/lyr? (Dry film thickness: about 20 fim)' (1) Fiber structure layer Fiber dispersions 1 to 6 having the composition shown in Table 5 below were coated on the above support and dried.

(54) (55) (2) Particle combination layer ((a) Uninvented exemplary compound (1) having an average particle size of 8 to 10μ
) containing a rabbit anti-α
- Fetoprotein antibody adsorption at 14 g/dm” (b) Nonionic surfactant Zeonyl FSN (E.

(2) (manufactured by DuPont) α014174m'' was adjusted to pH 7.2 with a phosphate buffer, and then an interactive composition-containing particle conjugate layer coated with the above composition was applied and dried.

The following test serum was applied to the immunoassay element for α-fetoprotein analysis of the present invention manufactured as described above. That is, for 10 μt of serum from which α-fetoprotein was removed from normal adult serum using an immunoatosorbent, 10 μt of fluorescently labeled α-fetoprotein was added as a labeled antigen and 0 mol was added as an unlabeled antigen.
(56) Prepare all the various α-fetoprotein test serums containing 10−5 mol of ~I×10−5, and drop 10 μt of each sound onto the immunoassay element of the present invention at 7? -0 10 μt of test serum was all absorbed into the interactive composition-containing particle conjugate layer within 15 seconds. Thereafter, the above element was incubated at 57°C for 20 minutes. Then add an excitation filter and an emission filter to each element f 490 nm and 515 nm. The fluorescence intensity of unbound labeled α-fetoprotein was measured across the entire polystyrene support using a reflection fluorometer equipped with a fluorometer. The results are shown in Table 4 (5
8) As shown in Table 4 above, the immunoassay element of the present invention is:
A complete immunoassay can be performed quickly by measuring the fluorescence corresponding to various concentrations of unlabeled α-fetoprotein contained in the test serum.

As explained in detail above, the analytical element of the present invention has excellent quantitative analysis accuracy, is superior to conventional analytical elements in terms of strength, and is remarkable in that it takes a very short time to accommodate the fluid sample in the layer. The effect is great.

Patent applicant Roku Konishi Photo Industry Co., Ltd. Agent Hirotoshi Nakamoto Akira Inoue (59) Procedural amendment (method) % formula % 1. Indication of case Patent application No. 125050 of 1981 2. Title of invention Immunoanalysis Material 5 correction? Relationship with the case involving the person who filed the patent application Patent applicant address: 1-26-2 Nishi-Shinjuku, Shinjuku-ku, Tokyo
Name (127) Konishiroku Photo Industry Co., Ltd. Representative Kawamoto Shinbashi Office 6-15-8 Nishi-Shinbashi, Minato-ku, Tokyo Nishi-Shimbashi Chuo-ku 02 Telephone (4!+7)-5467 Name
Patent attorney (7850) Hiroshi Nakamoto (and 1 others)
(Name) 5. Date of amendment order: October 7, 1980 (Shipping date: October 26, 1982) 6. Subject of amendment: Full text of the specification

Claims (1)

[Claims] 1. At least one fibrous structure layer and at least one particle combination layer containing a reactive group? Which components in the fluid sample have? In the analytical element to be analyzed, the particle combination layer has a porosity of 25 to 8 to enable transport of the fluid.
consisting of a particle assembly that is a non-swellable three-dimensional lattice with 5 interconnected voids, the particles being non-swellable and impermeable to the fluid and containing reactive groups,
An analytical element comprising thermostable organic polymer particles having a size of .mu., and wherein the fibrous structure layer is formed by coating a fiber dispersion with gold. Z The analytical element according to claim 1, wherein the thermally stable organic polymer particles are directly chemically bonded by the reactive group at the contact portion between the particles. The analytical element according to claim 1, wherein the organic polymer particles are chemically bonded by the reactive group via a low-molecular compound at the bonding portion between particle units.