JPH0672884B2 - Analytical element - Google Patents

Description

TECHNICAL FIELD The present invention relates to an analytical element for measuring a trace component in a fluid sample, and more particularly to an analytical element for analyzing a specific trace component in a biological fluid sample. .

[Conventional technology]

Various analytical methods have been developed as a method for detecting a substance contained in a trace amount contained in a biological fluid sample. The analysis method is based on the principle of immune reaction. Although various methods have been developed as the measurement method using the above principle, the most accurate method is as follows.
Immunoassays are known.

The immunoassay was successfully performed in 1958 by Berson and Yallow using serum iodine-labeled bovine insulin and anti-insulin antibodies in sera from patients with diabetes. Since then, radioimmunoassay has been widely used.

Since then, various labeling compounds other than radioisotopes have been developed. Other labeling compounds include, for example, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metal and organometallic complexes,
Examples include organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules.

As one of the important technical problems relating to the above-mentioned immunoassay, separation of a substance that causes binding (hereinafter abbreviated as B) and a substance that does not cause binding (hereinafter abbreviated as F) (hereinafter B / F) Abbreviated as separation).

Conventionally, various methods have been developed in order to solve the problems in the immunoassay method (for example, JP-A-53-38619).
No. 53-79024, No. 55-90859, No. 57-67860,
57-200862, 58-18167, 59-77356 and
59-170768).

However, these methods have drawbacks such as incomplete B / F separation, noisy signal reliability problems, and measurable substances limited to low molecular weight substances.

[Problems to be solved by the invention]

On the other hand, in Wet Chemistry, an immunoassay method by a competitive method using a stationary phase has been developed (see, for example, JP-A Nos. 58-209994 and 59-202064). However, in these measuring methods, the whole enzyme activity is measured without distinguishing both stationary phases, and therefore satisfactory results regarding sensitivity, accuracy and reproducibility cannot be obtained due to problems of background and noise.

On the other hand, in dry chemistry, an immunoassay method using a second antibody has been developed (see JP-A Nos. 57-82766 and 57-82767). However, there is room for improvement in these because of complicated operations and the need for a technique for developing with good reproducibility. Furthermore, JP-A-59-
The invention described in Japanese Patent No. 34155 discloses a method of using an unbound object storage sheet. However, even in this method, the above-mentioned problem occurs when the measurement is performed while the reaction sheet and the unbound material storage sheet are in close contact with each other, and it is complicated to separate both sheets at the time of measurement, and particularly the measurement is automated. It becomes an obstacle when doing.

The present invention has been made to improve the above-mentioned drawbacks of the prior art, and the purpose thereof is to perform B / F separation in a single layer to obtain a wide range of molecular weight (100
It is intended to provide an analysis element capable of quantifying a specific component in a fluid sample, which can be applied to substances of up to 5 million) and has excellent sensitivity, accuracy and reproducibility by a simple operation.

[Means for solving problems]

Briefly describing the present invention, the present invention relates to an analytical element, which is a substance B capable of specifically binding the specific component A in a fluid sample to the specific component A, and the specific component A or an analog thereof. And (a) a carrier on which the substance B is immobilized, and (b) the label which is not bound to the substance B, in an analytical element for measurement by a competitive reaction between the labeled substance C and a label C bound to the substance B. Substance D that specifically binds to the labeling site of substance C and modulates the signal caused by the label (hereinafter abbreviated as absorbing substance D)
It has a porous reaction layer formed by using a mixture containing a carrier on which is immobilized.

The fluid sample that can be used in the present invention can be any form of solution or colloidal solution, but preferably a biological fluid sample, that is, blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk, urine. , Sweat, gravy, etc., and particularly preferred are blood and serum.

The specific component A in the fluid sample that can be measured by the present invention is a substance or a group of substances whose presence or amount in the fluid sample can be measured to obtain a substance that specifically binds to the component,
The substances or groups of substances listed in Table 1 below may be mentioned by way of example.

The substance B that specifically binds to the specific component A in the fluid sample that can be used in the present invention includes an antibody, an antigen,
Examples include lectin, protein A, and an inhibitor of a specific enzyme, and it is particularly preferable that the binding reaction between the specific component and the binding substance is an antigen-antibody reaction. The antibody used in the present invention is not particularly limited in its origin, and an antiserum obtained by immunizing a mammal with an antigen, immunization, ascites fluid, or a conventionally known method (Right field) Shunsuke ed. “Immunochemistry”, Nakayama Shoten, pp. 74-88), sodium sulfate precipitation method, ammonium sulfate precipitation method, gel filtration method using Sephadex gel, ion-exchange cellulose chromatography method, electrophoresis method, etc. Can be used.

Alternatively, monoclonal cells may be prepared by obtaining hybrid cells (hypridoma) from spleen cells (eg, mouse) sensitized with an antigen and myeloma cells (myeloma).

Further, these antibodies may be IgG, IgM, IgA, IgD, IgE fractions, or may be used as active antibody fragments with Fab or Fab 'by enzymatically treating these antibodies. . Furthermore, these antibodies may be used alone or in combination of a plurality of antibodies.

When an antibody or an antigen is used as the substance B that specifically binds to the specific component A in the fluid sample, the measurement principle of the analytical element of the present invention belongs to the immunoassay method. The analytical element of the present invention can be particularly preferably used in an immunoassay, but is not limited to the immunoassay, and it is apparent from the contents described above that various applications are possible.

Labels applicable to the present invention include, for example, substances that change the activity of enzymes, enzyme substrates, enzymes and enzyme precursors (enzyme inhibitors, coenzymes, prosthetic groups, substances that activate enzyme precursors, etc.) , Enzyme precursors, apoenzymes, fluorescent substances and the like, and typical examples thereof include the substances shown in Table 2 below. More preferably, the enzymes and fluorescent substances disclosed in Table 2 are used. (The signals due to these signs will be described later.

2. Substrate p-Nitrophenyl-β-D-galactoside o-Nitrophenyl-β-D-galactoside 4-Methylumbelliferyl-β-D-galactoside p-Nitrophenylphosphonate Cortisol-21-hemisuccinate Umbelliferone Conjugate Luminol Iso Luminol N- (4-aminobutyl) -N-ethyl isorminol Hemisuccinamide N- (6-aminohexyl) -N-ethyl isorminol N- (4-aminobutyl) -N-ethyl isorminol Lucigenin Acrydinium Phenyl carboxylate Lofine pyrogallol Siloxin gallate Bis (2,4,6-trichlorophenyl) oxalate and its derivatives 3. Enzyme inhibitor Physostigmine Methionine Sulfoximine Wildfire poison Elementary blue dextran o-dianisidine-cellulose o-dianisidine-dextran 2-propynylamine 2-chloroallylamine phenylglycine p-nitrophenylglycine aminoacetonitrile 2-amino-3-hydroxypropyl-1,3′-carboxy-3 ′ - amino-1'-propenyl -1 ether L-2-amino-4-methoxy - trans-3-butenoic acid ethanolamine o- Sarufueto Arubijiin azaserine diazo-oxo-norleucine diazo oxo NOR-nor-valine △ ^{3-7-Aminosefuarosu} Polyacid Mimosine 2-Amino-4-pentynoic acid 2-Amino-4-chloro-4-pentenoic acid 3,3-Dichloroalanine 3,3,3-Trichloroalanine D-Cycloserine 2-hydroxyl-3-butyric acid N, N-trimethyl-2 Propynylamine β-aminopropionitrile 2-Promoethylamine 3-decinoyl-N-acetylcysteamine 2,3-decadienoyl-N-acetylcysteamine β-chloro-L-alanine L-serine-o-sulfate β-fluoroalanine L- Vinylglycine D-Vinylglycine Propargylglycine Gabacrine 5-Nitro-L-norparin N-Benzyl-N-methyl-2-propynylamine 3-Dimethylamino-1-propyne Glycerol Diisopropylphosphorofluoride Phenylmethanesulfonyl fluoride Clavulane Acid allopurinol 4. Coenzyme / prosthetic group FAD (flavin adenine dinucleotide) FMN (flavin mononucleotide) heme S-adenosylmethionine THF (tetrafudrofolate) TPP (thiami) Diphosphate) CoA (coenzyme A) UDP-Glc (uridine diphosphate glucose) PLP (pyridoxal phosphate) ATP (adenosine triphosphate) biotin CoI (nicotinamide adenine dinucleotide) CoII (nicotinamide adenine dinucleotide phosphorus) Acid) Adenosyl cobalamin Methyl cobalamin CoM (2,2'-dithiodiethane sulfonic acid) CoQ (ubiquinone) 5. Apoenzyme apoglutathione reductase apototechrome reductase apoNADPH dehydrogenase apoglucose oxidase apolipoamide dehydrogee Nase Apopyridoxine phosphate oxidase Apoperoxidase Apocytochrome C Apoxanthine oxidase Apo yeast lactate dehydrogenase Apo sarcosine oxidase Apo p-hydroxybenzoic acid hydroxylase Apoacy Le-CoA dehydrogenase Aposihydroriboacid dehydrogenase Apo-succinate dehydrogenase Apohomocysteine methyl transferase Apoglutamate formyl transferase Apotrans ketolase Apocholine acetyl transferase Apoglycogen synthase Apoalanine amino transferase Apohexokinase 6. Enzyme precursor Substances that activate the body Enteropeptidease Streptokinase Protein kinases Enzyme precursors Various proteases 7. Enzyme precursors trypsinogen chymotrypsinogen procolipase prophospholipase prorenin procarboxypeptidase A procarboxypeptidase B quininogen proelastase angiotensinogen proinsulin proparati Lloyd Lumon Proglucagon Procollagen (Soluble) Aggregation factors XI, XII, XIII Procollagenase Prococornase Prekallikrein Pepsinogen Plasminogen Fiuprinogen prothrombin Plasminogen proactivator Proacrodin 8. Fluorescent substance Fluorescein isothiocyanate (FITC) Tetramethylrhodamine Isothiocyanate (TRIT
C) Rhodamine B Isothiocyanate (RBITC) Lissamine Rhodamine-B200 Sulphonyl chloride (RB
200SC) Umbelliferone 4-Methylumbelliferone (4MU) Fluorescein Ophthalvamil (FTC) Fluorescein Thiocarbamyl-diphenylglycine (FTC-DPG) Tetramethylrhodamine (TMR) 5-[(4,6-Dichlorotriazine-2 -Yl) -amino] fluorescein dimethylaminonaphthalene-5-sulfonyl chloride (DNS-Cl) fluoram 2-methoxy-2,4-diphenyl-3 (2H) -furanone (MDPF) 7-chloro-4-nitrobenzo-2- Oxa-1,3-diazole (NBD-Cl) 1-anilino-8-naphthalenesulfonic acid (ANS) N- (3-pyrene) -maleimide (NPM) N- (7-dimethylamino-4-methyl-2- Oxy-
3-Chloromethyl) -maleimide (DACM) N- (p-2-benzimidazolyl-phenyl) -maleimide (BIPM) Anthracene isothiocyanato fluoroantirumaleimide (FAM) Various chelates containing rare earth elements and their derivatives Specific components Alternatively, a labeled substance C in which an analog thereof and a label are bound to each other
(Hereinafter, abbreviated as “labeled substance C”) means that the substance B capable of specifically binding to the above-mentioned specific component A can be specifically bound in the same manner as or similar to the specific component, and It is a generic term for substances that are directly or indirectly bound by chemical means while retaining the ability to emit signals. Actually, it can be obtained by chemically binding the above-mentioned label to a substance having an antigenic determinant common to the specific component or the specific component by a known method, and more specifically, Eiji Ishikawa,
Kawai Tadashi, Miyagawa Kiyoshi "Enzyme Immunoassay (2nd Edition)"
(Medical Shoin, published in 1978) and Japanese Society for Clinical Pathology "Clinical Pathology" Extra edition special issue No. 53 "ImmunoAssay for clinical examination-Technology and application-" (Clinical Pathology Publishing, 1983) Can be used as a reference. Among these methods, the glutaraldehyde method, the periodate acid method (Nakane method), and the maleimide method can be particularly preferably used.

The absorbing substance D used in the present invention, that is, the substance D that specifically binds to the labeling site of the precursor labeled substance C to eliminate or reduce the signal caused by the label is selected according to the label used. It should be done, and the following substances can be mentioned as examples.

1. When label is “enzyme” Signal due to label Decrease in substrate due to the enzyme activity, increase in product, emission of energy and changes caused by them, preferable absorbing substance D Inhibitor for the enzyme (Table 2 Those that correspond to the enzyme can be selected and used from the inhibitors listed in 1.) Antibodies to the enzyme that bind to the enzyme and affect its activity 2. When the label is an "enzyme substrate" Signal due to increase in product caused by reaction of the substrate with an enzyme added in the analytical element, emission of energy and change caused by them, preferable absorbing substance D binding to substrate with antibody to the substrate That inhibits the enzyme reaction.

Enzyme that incorporates the substrate as an irreversible inhibitor An enzyme that uses the substrate as a substrate and does not give the signal that is originally to be detected by the reaction.

3. When the label is "coenzyme" or "prosthetic group" Signal due to label Reduction of substrate / increase of product due to reaction of enzyme added to the analytical element that requires the label, and those Preferred absorption substance D An antibody against the label, which binds to the label and affects its activity, or which absorbs or consumes the label but does not give a signal originally intended to be detected by the activity .

4. When the label is "apoenzyme" Signal originating from the label The label does not generate a signal as it is. It is possible to measure the decrease in the substrate, the increase in the product, and the change caused by the expression of the enzyme activity by binding to the absorbing substance described below.

Preferred Absorbent D A prosthetic group that expresses the enzyme activity of the label. (A corresponding one of the prosthetic groups shown in Table 2 can be selected and used.) 5. When the label is a “substance that activates a zymogen” A signal originating from the label Activates the zymogen added therein, decreases the substrate due to the activity, increases the product, and changes caused by them, preferable absorbing substance D An antibody against the substance is bound to the substance and its activity is increased. What has an impact.

When the substance is an enzyme, its inhibitor 6. When the label is a “enzyme precursor” Signal originating from the label The label does not give a signal as it is. A part of the molecule is cleaved after binding to the absorbing substance described later, and the enzyme activity is expressed, and the decrease in the substrate, the increase in the product, and the resulting change can be measured.

Preferred absorbing substance D A substance that expresses the enzymatic activity of the label 7. When the label is a "fluorescent substance" Signal originating from the label Fluorescence emitted when the fluorescent substance is irradiated with excitation light, preferable absorbing substance D An antibody or derivative thereof that changes the fluorescence wavelength and intensity of the label.

Although specific examples of the above-mentioned various absorbing materials are well known to those skilled in the art, and needless to disclose them again, representative examples will be shown below in order to facilitate understanding of the present invention.

Examples of combinations of enzymes and inhibitors that can be used in the present invention include biotin enzymes (pyruvate carboxylase, acetyl Co).
-A carboxylase, propionyl-CoA carboxylase, methylmalonyl-CoA carboxylase, etc.) and apidine, peroxidase and o-dianidin-dextran, lactate oxidase and 2-hydroxyl-3-butyric acid, monoamine oxidase and N, N-trimethyl-
2-propynylamine, β-aminopropionitrile, etc. are mentioned, and further, Journal of the American Chemical Society (J. Am. Chem. Soc) No. 80
Volume, p. 456 (1958); Vol. 82, p. 596 (1960).
Years); Accounts of Chemical Research (Aco.Ch
em.Res) Volume 9, 313 (1976); Science (Scien)
ce) Volume 185, page 320 (1974); Chemical Industry 1985, page 21 (19)
The enzyme / inhibitor combinations described or cited in (1985) and the like can also be preferably used.

The porous reaction layer forming the main part of the present invention is formed (for example, by coating and / or film forming) using a mixture containing the carrier represented by the above (a) and (b), Examples of the carrier include particles and fibers.

The particles used here preferably have a particle size of 1 to 1000 μm, and examples of the material include dextran, agarose, polyamide,
Polystyrene, polycarbonate, or JP-A-55-
Organic polymer particles such as those disclosed in Japanese Patent No. 90859, or inorganic particles such as glass, silicon dioxide and silica sand can be preferably used.
It is particularly preferable to use the self-bonding particles disclosed in JP-A Nos. 60 and 57-101761. Examples of typical materials include those shown in Table 3 below.

Table 3 Exemplified compounds (1) Poly (stilne-co-glycidyl methacrylate) [90/10].

(2) Poly (styrene-co-methyl acrylate-co-glycidyl methacrylate) [80/15/5].

(3) Poly (styrene-co-n-butylmethacrylate-co-glycidylmethacrylate) [75/15/10].

(4) Poly (styrene-co-vinylbenzyl chloride-co-glycidyl methacrylate) [80/10/10].

(5) Poly (styrene-co-divinylbenzene-co-
Glycidyl acrylate) [90/2/8].

(6) Poly (p-vinyltoluene-co-glycidyl methacrylate) [90/10].

(7) Poly (methacrylate-co-glycidyl methacrylate [80/20].

(8) Poly (styrene-co-N, N-dimethylaminoethyl methacrylate) [95/5].

(9) Poly (styrene-co-aziridylethylmethacrylate) [95/5].

(10) Poly (styrene-co-methyl acrylate-co-acrolein) [90/5/5].

(11) Poly (styrene-co-acrylamide) [95 /
Five〕.

(12) Poly (styrene-co-vinylthiol) [95 /
Five〕.

(13) Poly (styrene-co-methylol acrylamide) [95/5].

(14) Poly (styrene-co-t-butyl acrylate-glycidyl methacrylate) [95/5/5].

(15) Poly (styrene-co-vinyl isocyanate)
[95/5].

(16) Poly (methyl acrylate-co-styrene-co-N-methylol acrylamide) [50/35/15].

(17) Poly (styrene-co-glycidyl methacrylate-co-N, N-dimethylaminoethyl methacrylate)
[90/5/5].

(18) Poly (styrene-co-methacrylic acid-co-acrylamide) [95/2/3].

(19) Poly (styrene-co-N-methylolacrylamide-methoxyethyl acrylate co-acrylate) [95/5/5].

(20) Poly (p-vinyltoluene-co-N-methylolacrylamide-co-acrylic acid) [95/8/2].

(21) Poly (methyl methacrylate-co-glycidyl methacrylate-co-t-butyl acrylate) [80/1
0/10].

(22) Poly (styrene-co-p-vinylbenzyl chloride-co-acrylic acid-co-ureidoethyl acrylate) [75/10/5/10].

(23) Poly (styrene-co-methacrolein-co-α
-Hydroxyethyl methacrylate) [90/5/5].

(24) Poly (styrene-co-acrolein-co-acetoacetoxyethyl methacrylate) [85/5/10].

(25) Poly (styrene-co-N, N-dimethylaminoethyl acrylate-co-vinylsulfonylethyl methacrylate) [90/5/5].

(26) Poly (p-vinyltoluene-co-aminostyrene-co-vinylsulfonylethyl methacrylate) [85
/ 10/5].

(27) Poly (styrene-co-N, N-dimethylaminoethyl methacrylate) [90/10].

(28) Poly (styrene-co-acrylic acid) [97/3].

(29) Poly (styrene-co-acrylamide) [97 /
3].

(30) Poly (p-vinyltoluene-co-t-butyl acrylate) [95/5].

(31) Poly (methyl acrylate-co-methacrylamide) [95/5].

(32) Poly (styrene-co-N-methylolacrylamide) [95/5].

(33) Poly (p-vinylbenzyl chloride-co-N
-Methylol acrylamide) [96/4].

(34) Poly (styrene-co-itaconic acid) [98/2].

(35) Poly (styrene-co-t-butyl acrylate) [92/8].

(36) Poly (methyl acrylate-co-styrene-co-acrolein) [30/65/5].

(37) Poly (methyl methacrylate-co-styrene-
Co-2-hydroxyethyl methacrylate) [25/70 /
Five〕.

(38) Poly (styrene-co-vinylsulfonylethyl acrylate) [80/20].

(39) Poly (styrene-co-N, N-dimethylaminoethyl acrylate) [90/10].

(40) Poly (styrene-methyl acrylate-co-acetoacetoxyethyl acrylate) [90/5/5].

(41) Poly (styrene-co-methacrylic acid) [9/5 /
Five〕.

The weight% of the monomer used in the polymerization reaction is shown in parentheses after each exemplified compound.

Alternatively, several kinds of these particles may be mixed and used.

The fibers used in the porous reaction layer of the present invention include valves, cotton, hemp, silk, wool, cellulose esters, viscose rayon, copper ammonia rayon, polyamide (6
-Nylon, 6,6-nylon, 6,10-nylon, etc.),
Polyester (polyethylene terephthalate, etc.), polyolefin (polypropylene, vinylon, etc.), glass fiber, asbestos, and other plant, animal, mineral, synthetic, semi-synthetic, regenerated fibers can be used, or a mixture of these can be used. May be.

Immobilization of a substance that specifically binds to the specific component can be achieved by physically adsorbing the substance on the surface of the above-mentioned particles or fibers by various known methods, or by directly or indirectly binding by a chemical reaction. It is achieved by At this time, it is necessary to pay attention not to lose the specific binding property of the substance to the specific component, for example, Eiji Ishikawa, Tadashi Kawai,
Kiyoshi Miyai "Enzyme Immunoassay (2nd Edition)" (Medical Shoin,
1978), Ichiro Chibata, Tetsuya Tosa, Yuji Matsuo, "Experiments and Applications Affinity Chromatographies" (Kodansha, 19
The method described in 1976) can be mentioned as an example of a preferable method.

In addition, after immobilizing the substance B that specifically binds to the specific component A, a protein that does not participate in the immune reaction to be measured is carried for the purpose of eliminating a nonspecific reaction in the immune reaction, if necessary. Is possible. Typical examples thereof include normal mammalian serum proteins, albumin, gelatin, and degradation products thereof.

The above (a) and (b) are prepared by the method described above. At this time, (a) and (b) do not necessarily have to be the same material, and particles of different materials, fibers of different materials, or particles and fibers may be used in combination. However,
In order to uniformly mix (a) and (b), it is not preferable to combine particles having significantly different average particle sizes.

The mixing ratio of the particles or fibers (a) and (b) is determined according to the amount of each immobilized specific binding substance and the specific number of binding, and if necessary, further fixing the specific binding substance. Unmodified particles or fibers may be added for conditioning.

By applying and / or forming a film of these mixtures,
Form a porous reaction layer in which there is a porous structure with interconnecting pores that can freely contact the fluid sample. Particles having no self-bonding property can be formed into a film in which the particles are spot-bonded to each other by using an appropriate adhesive, for example, JP-A-55-90.
The method of Japanese Patent No. 859 can be applied. Organic polymer particles having self-bonding property are disclosed in JP-A-57-101760 or
A film can be similarly formed by the method described in each of Japanese Patent Publication No. 57-101761. For fibers or mixtures of fibers and particles, see JP-A-57
A porous reaction layer can be formed by applying the fiber dispersion described in JP-A-125847 and JP-A-57-197466. It is also possible to apply a fiber dispersion liquid using a water-soluble binder such as gelatin or polyvinylpyrrolidone, as in the method described in Japanese Patent Application No. 59-28571.

The form of the analytical element of the present invention is not particularly limited as long as it can be analyzed, but is preferably in the form of a film or a sheet in terms of production and operation, measurement.

In order to help understanding of the analytical element of the present invention, the principle will be described with an example.

FIG. 1 is an example of a cross-sectional view of the simplest mode of an analysis element according to the present invention. In this case, the analysis element is composed of only the porous reaction layer 1.

FIG. 2 is an enlarged view of FIG. Reference numeral 2 in FIG.
Are particles of (a), 3 are particles of (b), and 4 are particles added for adjustment.

FIG. 2 shows that particles 2, 3 and 4 are uniformly mixed and point-bonded to form a porous reaction layer.

FIG. 3 is a schematic view of the labeled substance C, where reference numeral 8 is a specific component in the fluid sample or its analog, 9 is a label, and 10 is the labeled substance C.

FIG. 4 is a schematic diagram for explaining the present invention. Reference numeral 5 is a substance B that specifically binds to a specific component in a fluid sample, 6 is an absorbing substance D, and 7 is a specific component A in the fluid sample. .

The mechanism of the present invention will be described with reference to FIGS.

First, a fixed amount of a fluid sample is weighed, mixed with the fixed amount of the above-mentioned labeled substance, and dropped on the analytical element of FIG.

The specific component A in the fluid sample and the added label C competitively react with the “substance that specifically binds to the specific component in the fluid sample” B immobilized on the surface of the particle 2 in FIG. To do.
Further, the labeled substance C is the absorbing substance D immobilized on the surface of the particle 3.
Also reacts. (FIG. 4) The labeled substance C after a certain reaction time is a substance B that specifically binds to the specific component A in the fluid sample.
There are three states, i.e., that is adsorbed to the particles on which is immobilized via the substance, b is adsorbed on the particles on which the absorbing substance D is immobilized via the substance, and remains in the fluid sample. Of these, it is preferable to set the reaction conditions and the constitution of the porous reaction layer so that the proportion of C is as small as possible. The ratio of B is controlled by the result of the competitive reaction between the specific component A and the labeled substance C in the fluid sample.
The higher the concentration of, the lower the ratio of A and the higher the ratio of B. In the labeled product in the state of (2), the signal due to the label is modulated by the binding between the labeled site and the absorbing substance D.
A functional relationship is established between the concentration of the specific component A in the fluid sample and the signal intensity of the entire label. Therefore, if a calibration curve is created using several types of fluid samples (standard samples) in which the concentration of the specific component A is known in advance, the concentration of the specific component in the unknown fluid sample can be known.

The method of measuring the signal depends on the type of sign. For example, when the label is a fluorescent substance, excitation light may be applied to the analysis element to measure the fluorescence intensity. If the label is an enzyme, a suitable substrate,
If necessary, add a solution containing an enzyme or a coloring system and incubate for a certain period of time, then reflect the light at a wavelength compatible with the coloring system (fluorescence intensity, color intensity depending on the type of substrate)
The signal strength can be measured by measuring As the substrate / color-developing system used for such a purpose, an appropriate one can be selected from known methods according to the kind of the labeling enzyme.

As an example, when the label is peroxidase, a buffer solution containing 0.001 to 10.0% hydrogen peroxide (pH 4.0 to 9.
It is possible to use one obtained by dissolving one or several of the compounds exemplified in Table 4 in (0).

Table 4 (1) o-dianidine (2) o-tolidine or an acid salt thereof (3) o-phenylenediamine or an acid salt thereof (4) guaiac (5) adrenaline (6) phenolphthalein (7) ferrocyanide (7) 8) Combination of 4-aminoantipyrine and its derivative or acid salt thereof with phenol or naphthol or their derivative (9) Aniline and its derivative (10) Monoamines such as o-toluidine and p-toluidine ( 11) o-phenylenediamine, N, N'-dimethyl-
Diamines such as p-phenylenediamine, N, N'-diethylphenylenediamine, benzidine, and dianisidine (12) phenol, thymol, o-, m-, and p-
Phenols such as cresol, α-naphthol and β-naphthol (13) Polyphenols such as catechol, guaiacol, orcinol, pyrogallol, p, p-dihydroxydiphenyl, phloroglucinol (14) Salicylic acid, pyrocanicins, gallic Aromatic acids such as acids (15) Leucomalachite green, leuco dyes such as leucophenolphthalein (16) Colored dyes such as 2,6-dichlorophenolindophenol (17) Epinephrine, flavones, tyrosine, Various biochemicals such as dihydroxyphenylalanine and tryptophan (18) 2,2'-azinodi (3-ethyl-6-sulfobenzothiazoline) or its salt, and special such as 3,3'-diaminobenzidine Dye (19) Others, Guaya gum, Guayaconic acid, Potassium iodide Substance, sodium iodide and other water-soluble iodides, and substances such as bilirubin whose label is an enzyme substrate, a coenzyme, an apoenzyme, a substance which activates a zymogen, or an enzyme. For example, a solution of a substance required for signal measurement may be added, the solution may be incubated, and the reflection density, fluorescence intensity, emission intensity, etc. may be measured.

The particles or fibers of the carrier shown in the above (a) and (b) used in the present invention form a reaction layer in a state where their surfaces are apart from each other, except for a slight adhesive portion when viewed microscopically, This secures a space for accommodating the fluid sample, and at the same time, the labeling site of the labeling substance C adsorbed to the substance B immobilized on the surface of the particle or fiber of (a) has the surface of the particle or fiber of (b). The signal is prevented from being modulated by the influence of the substance immobilized on the. As a result,
B / F separation within a single layer, which is a feature of the present invention, has become possible.

Furthermore, as another effect of the present invention, when mixing the particles or fibers of (a) and (b) and particles or fibers added for adjustment as necessary, the ratio can be freely set, Amount of substance immobilized on (a) and (b) and labeled substance C
Even if the binding ability to γ varies slightly depending on the manufacturing lot, it is possible to supply an analytical element having a constant performance by adjusting the mixing ratio. Conventionally, an analytical element and a similar product utilizing a porous layer of particles or fiber aggregates handle an aggregate of particles or fibers simply as a container for storing a fluid sample, and particles constituting such an aggregate,
It is a completely new attempt to perform B / F separation in a single layer by mixing fibers with different functions to form a film, and the inventors were able to obtain the above-mentioned effects. It was a surprising result that was completely unexpected.

In the analytical element of the present invention, the above-mentioned porous reaction layer is the minimum necessary constituent element, but various auxiliary layers can be provided to further exert the effects of the present invention. 5 to 10 are schematic sectional views showing an embodiment of the analytical element of the present invention.

In the analytical element of the present invention shown in FIG. 5, the porous reaction layer 1 is laminated on the light transmissive support 11, and the presence of the support improves the handleability of the element. The support which can be used for such a purpose includes, for example, polymer compounds such as cellulose acetate, polyethylene terephthalate, polycarbonate and polyvinyl compounds (eg polystyrene),
Alternatively, a transparent inorganic compound such as glass may be used.
The porous reaction layer may be directly applied and / or formed into a film on such a support, or may be formed on the support after forming a separate porous reaction layer. Fifth
In the case of the embodiment shown in the figure, the fluid sample needs to be dropped from the reaction layer side, but the signal can be measured from both sides.

In another embodiment of the invention shown in FIG. 6, a light reflective support is provided.
A reaction layer is provided on the surface 12. In this embodiment, both the sample dropping and the signal measurement are performed from the porous reaction layer side, and the light reflecting support facilitates the measurement of the signal with the reflection density. (A similar effect can be obtained by similarly using a black absorptive support when measuring the signal by fluorescence intensity).

As the material of the support that can be used for such a purpose, in addition to the above-mentioned material of the support, ceramics, metal, or paper waterproofed with a resin coating or the like can be used. The purpose can be achieved by applying or containing a white pigment such as TiO ₂ , BaSO ₄ , or mica.

The embodiment shown in FIG. 7 has the same purpose, and the porous reaction layer 1 and the light reflecting layer 13 are sequentially laminated on the light transmitting support 11. In this mode, the sample is dropped from the light reflection layer side,
The signal measurement is performed from the side of the light transmissive support. The light-reflecting layer may be any of those used in known analytical elements and similar products, but preferably the same particles and fibers as those used in the porous reaction layer are allowed to contain the aforementioned white pigment and the like. It can be applied, formed into a film, or attached. More preferably, the substance B or the absorbing substance D which specifically binds to a specific component in the fluid sample is immobilized on the surface of the particles containing the white pigment in the porous reaction layer, and the lower layer of the porous layer is made porous. A light reflection layer having the same function as the reaction layer can be provided. The use of such a special light reflecting layer can prevent a large amount of unreacted labeling substance from remaining in the light reflecting layer.

In the embodiment shown in FIG. 8, the labeling substance-containing layer 14 is formed on the porous reaction layer 1.
Is provided. The labeled substance-containing layer is a layer in which the labeled substance is contained in the porous medium so that the area concentration becomes constant, and when a fixed amount of the fluid sample is dropped, a fixed amount of the labeled substance is eluted and the sample is placed in the porous reaction layer. It diffuses with. As the material, the same material as the porous reaction layer may be applied, film-formed, or attached, or water-absorbent paper, Japanese paper, paper, brush polymer, or glass fiber / mineral fiber (asbestos), plant Sex fibers (cotton, hemp, pulp, etc.),
Woven fabrics, non-woven fabrics, synthetic papers, etc. made of animal fibers (wool, silk, etc.), synthetic fibers (various nylons, vinylon, polyethylene terephthalate, polypropylene, etc.), recycled fibers (rayon, cellulose ester, etc.) alone or mixed. The labeled substance-containing layer prepared in step 1 may be attached. If necessary, the marker-containing layer can also have the effect of the light reflecting layer described above. In the case of the embodiment of FIG. 8, if the label is a fluorescent substance, only the fluid sample is dropped,
Immediate measurement is possible by incubating. When the label is another substance, it can be measured by dropping a fluid sample for a certain period of time and then dropping a solution containing a necessary substrate, a coloring reagent and the like.

FIG. 9 also shows another embodiment having a marker-containing layer. In this case, the light transmissive support 11, the marker-containing layer 14, the timing layer 15, and the porous reaction layer 1 are laminated in this order from the bottom.

The timing layer is a technique widely known in the field of photographic chemistry, and for example, a gelatin layer having an appropriately controlled degree of hardening is used. In this embodiment, after the fluid sample is dropped, the specific component in the sample is adsorbed to the surface of the corresponding particles in the porous reaction layer to some extent, and then the labeling substance is released to the porous reaction layer. Such a method is widely known as a twisted decision and is effective in detecting a trace amount of components.

When the labeled substance-containing layer is in a portion other than the uppermost layer as in the present embodiment, the materials used for the labeled substance-containing layer include gelatin, gelatin derivatives, polysaccharides (such as agarose), carboxymethyl cellulose, and hydroxyethyl cellulose, in addition to the materials described above. There is a method in which a hydrophilic polymer substance such as, or a homopolymer or copolymer having vinylpyrrolidone, an acrylic acid derivative, a methacrylic acid derivative, vinyl alcohol, sulfonylstyrene, etc. as a monomer and a continuous binder are applied and used. As yet another aspect, by adjusting the film thickness and composition of such hydrophilic polymer or polymer, it is possible to control the elution rate of the labeled substance from the labeled substance-containing layer and also serve as the timing layer. .

The embodiment shown in FIG. 10 is particularly useful when the label is other than a fluorescent substance (in the case of a label that utilizes an enzymatic reaction in some form for signal measurement). The reagent layer 16 is the labeling substance-containing layer 14
It is a material similar to the above, and contains a substance necessary for the measurement of the labeling signal at a constant area concentration. By providing this reagent layer and the labeling substance-containing layer, even when a label other than the fluorescent substance is used, it is possible to measure by simply dropping only the fluid sample and incubating.

In this case, it is preferable that the content of the reagent layer is eluted after the reaction in the porous reaction layer has sufficiently proceeded. For that purpose, the above-mentioned timing layer is provided on the reagent layer or the composition of the reagent layer is Must be adjusted to have the effect of the timing layer. For the same reason, the reagent layer is preferably provided on the lowermost layer of the analytical element (on the support, if any).

When using peroxidase in the enzyme reaction system,
Hydrogen peroxide and a suitable reducing substance are required as substrates.
Of these, the former is volatile and it is difficult to incorporate it into the device as it is.Therefore, either contain it in the form of cumene / H ₂ O ₂ or generate hydrogen peroxide when the fluid sample is dropped. Preferably. For the latter, does an oxidase represented by glucose oxidase, urea oxidase, and amine oxidase (a type that produces hydrogen peroxide as a reaction product) and a substrate for the enzyme are contained in the reagent layer in a dry state? Alternatively, one of the oxidase and the substrate may be contained in a reagent layer and the other may be contained in a different layer.

11 and 12 show a sectional view and a perspective view of an example of a preferred embodiment of the present invention. To facilitate the handling of the analytical element, the whole is covered with a plastic mount 17, a sample injection hole is formed in the upper part of the mount, and a signal measurement hole is formed in the lower part.

The analysis element of the present invention further comprises a spreading layer that assists the development of the fluid sample when it is applied to the element, and the fluid sample is blood (whole blood).
In this case, a blood cell separation layer, which may be necessary, an adhesive layer provided if necessary, a protective layer and an auxiliary layer can be provided. The auxiliary layer, the color-developing reagent layer, the label-containing layer, and the timing layer described above may be provided independently, or may be provided as a layer having a plurality of functions. The positions of these layers to be provided can be easily determined according to their functions.

〔Example〕

Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of rabbit anti-FITC antibody 50 mg of bovine serum albumin (BSA) was dissolved in 5 ml of 0.1 molar sodium carbonate solution (pH 9.0), and 2 mg of fluorescein isothiocyanate (manufactured by Research Organics, Inc., USA) was prepared. )
Was dissolved in 500 μl of dimethylformamide, added, stirred for 3 hours in the dark at room temperature, purified on a Sephadex G-25 column (manufactured by Pharmacia) and lyophilized.

This was mixed with Freund's complete adjuvant (first time only) and incomplete adjuvant to immunize rabbits.

The globulin fraction was separated from the obtained antiserum by the ammonium sulfate method, and BSA was performed by affinity chromatography.
After removing the antibody adsorbed on, the mixture was dialyzed and freeze-dried.

(2) Preparation of porous reaction layer (a) 0.05 M p of goat anti-human IgG (manufactured by Katsube Co., USA)
H9.6 Poly (styrene-co-n-butylmethacrylate-co-glycidyl acrylate) with an average particle size of 21 μm dissolved in carbonic acid / bicarbonate buffer at a concentration of 10 μg / ml [75/15/10]
The polymer polymer particle unit was added and left at 4 ° C. overnight.
The particles were washed with physiological saline, then put in the same buffer containing 200 μg / ml of BSA, left at 4 ° C. for 3 days, and washed with physiological saline.

(A) The same operation as (a) was performed using the anti-FITC antibody prepared in (1) instead of anti-human IgG.

(C) Particles in which only BSA was physically adsorbed were prepared by the method according to (A).

The particles prepared in the above (a) to (c) were uniformly mixed at a ratio of 4: 1: 5, and dried to a thickness of about 5% by weight with Triton X-100 (Nonionic surfactant, manufactured by Rohm Endhurth).
It was coated on a polyethylene terephthalate support so as to have a thickness of 350 μm, dried at 42 ° C. for 30 minutes, and peeled from the support after film formation.

(3) Preparation of Labeled Material-Containing Layer A reagent layer having the following composition was applied and dried on a subbed polyethylene terephthalate film.

(4) Preparation of Analytical Element The porous reaction layer prepared in (2) was adhered on the label-containing layer prepared in (3), and cut into a size of 2 × 2 cm to obtain an analytical element.

(5) Measurement of human IgG Human IgG (manufactured by Katsuberu USA) from 640 μg / ml to 0 μg / ml
0.01M sodium phosphate buffer solution (p
H7.6) is created.

When 10 μl of human IgG solution of each concentration was dropped on each one of the analytical element prepared in (4) and kept at 37 ° C. for 20 minutes, and then the fluorescence was measured from the sample dropping side (excitation wavelength 490 nm, fluorescence wavelength 520 nm). The results shown in Table 5 were obtained.

Example 2 (1) Preparation of Reagent Layer A color forming reagent layer having the following composition was applied onto a film of subbed polyethylene terephthalate and dried.

(2) Preparation of label-containing layer The following label-containing layer was applied onto the reagent layer prepared in (1) and dried.

(3) Introduction of Reactive Group into Cellulose Fiber 55 g of powdered paper D (Toyo Paper Co., Ltd.) was immersed in 1000 ml of 2.5 M potassium phosphate buffer (pH 12.1) and stirred with a stirrer at 5 to 10 ° C. While adding 500 ml of Promocyan solution (0.05 g / ml) gradually, the mixture was reacted for 20 minutes, passed, and washed successively with ice-cooled distilled water and 0.1 M sodium hydrogen carbonate.

(4) Preparation of porous reaction layer (a) Goat anti-human IgG (manufactured by Katsubel, USA) IgG fraction 1.0 mg (converted to antibody protein amount) was added to 40 ml of 0.1 M NaHCO 3.
_It was dissolved in a 3-0.5 M NaCl solution, a part of the activated paper prepared in (3) was added, and the mixture was shaken at room temperature for 3 hours. After the 1M
Shake with Tris-HCl buffer (pH 8.0) at room temperature for 2 hours. Water, 0.5M acetate buffer (pH 4.0), 0.5M NaHCO ₃ , 50 mM
Wash sequentially with phosphate buffered saline (pH 7.2). Add this to 50 mM Tris-HCl buffer (pH 8.0) containing 2% BSA.
It was soaked in the evening, washed with distilled water, contained a small amount of an aqueous solution of BSA and sucrose, and then freeze-dried.

(A) A part of the activated paper prepared in (3) was allowed to react overnight at room temperature in the dark at room temperature. After 1M
Treated with 1-amino-2-propanol and washed with water (A)
It was treated with BSA in the same manner as above and dried.

(C) The same operation as (a) was performed using glucose oxidase and a part of the activated paper prepared in (3).

As described above, a fiber dispersion having the following composition was prepared using the three types of treated fibers, and applied and dried on the marker-containing layer prepared in (2).

After drying, it was cut into a size of 2 × 2 cm to obtain an analytical element.

(5) Measurement of human IgG Human IgG (manufactured by Katsuberu USA) from 640 μg / ml to 0 μg / ml
0.01M sodium phosphate buffer solution (p
H7.6) is created.

10 μl of human IgG solution at each concentration was dropped on each one of the analytical elements prepared in (4), and after keeping the temperature at 37 ° C. for 20 minutes, the reflection density at 492 nm from the support side was measured. The results are shown in Table 6.

[Effects of the Invention] As described above, according to the analysis element of the present invention, B / F separation is performed in a single layer, and sensitivity, accuracy and reproducibility are improved by a simple operation, and a wide range in a fluid sample is obtained. It is possible to exert a remarkable effect that a specific component having a molecular weight can be quantitatively analyzed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 and FIGS. 5 to 10 are schematic sectional views showing an embodiment of the analytical element of the present invention, FIG. 2 is an enlarged view of FIG. 1, and FIG. FIG. 4 is a schematic diagram for explaining the present invention, FIG. 4 is a schematic diagram for explaining the present invention, FIG. 11 is a sectional view of an example of a preferred embodiment of the present invention, and FIG. 12 is a perspective view of FIG. 1: Porous reaction layer, 2: Carrier (a), 3: Carrier (b) 4: Particle, 5: Substance B, 6: Absorbing substance 7: Specific component A, 8: Label, 10: Labeled substance C 11: Light-transmissive support, 12: Light-reflective support 13: Light-reflecting layer, 14: Labeled substance C-containing layer 15: Timing layer, 16: Reagent layer, 17: Mount

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masayo Ishikawa 1 Sakura-cho, Hino City, Tokyo Konishi Roku Photo Industry Co., Ltd. (56) References JP-A-58-17365 (JP, A) JP-A-57 -103055 (JP, A) JP-A-58-18167 (JP, A)

Claims (1)

[Claims] 1. A specific component A in a fluid sample
In a analytical element for measuring by a competitive reaction between a substance B capable of specifically binding with and a labeled substance C in which the specific component A or its analog and a label are bound, (a) the substance B An immobilized carrier and (b) a carrier on which a substance D that specifically binds to a labeling site of the labeled substance C that is not bound to the substance B and modulates a signal caused by the label is immobilized. An analytical element having a porous reaction layer formed by using a mixture containing the element.