JPS6290539A - Analytical element - Google Patents

Abstract

PURPOSE:To obtain an analytical element for quantifying a specific component excellent in sensitivity, accuracy and reproducibility, by simple operation such that a porous reaction layer is formed of a mixture containing a carrier, which immobilized a substance specifically bonding a specific component, and a carrier which immobilized a substance specifically bonded to the labelled site of a predetermined labelled substance to modulate a signal. CONSTITUTION:In an analytical element for measuring the specific component A in a fluid specimen using a substance B capable of specifically bonding the specific component A, a substance C different from the substance B and capable of specifically bonding the specific component A and a labelled substance D having a marker bonded thereto, a porous reaction layer is formed by using a carrier which immobilized a substance A and a carrier which immobilized a substance E specifically bonded to the labelled site of the labelled substance D to modulate a signal caused by the labelled substance. In this immobilizing operation, the porous reaction layer may be formed after said substance are preliminarily immobilized by a particulate material or a fiber or immobilizing operation may be performed after the porous reaction layer was formed.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analytical element for measuring trace components in a fluid sample, and particularly to an analytical element for analyzing a specific component in a biological fluid sample. 〇 [Prior art] Regarding analytical elements Various analytical methods have been developed as methods for detecting trace amounts of substances contained in biological fluid samples. The analysis method is mainly based on the immune reaction. Although various measurement methods have been developed using the above principle, immunoassay is known as the most accurate method.

The immunoassay method was developed in 1958 by Persson (Ber-8on).
) and YalloW succeeded in measuring insulin in serum using radioactive iodine-labeled bovine insulin and anti-insulin antibodies in the serum of diabetic subjects. Radioimmunoassay has been widely used since then. It is used.

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

One of the important technical problems regarding the above immunoassay is the separation of the substance that caused binding (hereinafter abbreviated as B) and the substance that did not bind (hereinafter abbreviated as F) (hereinafter referred to as B/
? (abbreviated as separation).

Conventionally, various methods have been developed to solve problems in immunoassay methods (for example, Japanese Patent Application Laid-Open No. 53-386
No. 19, No. 53-79024, No. 55-90859, No. 57-67860, No. 5 Fu 200862, No. 58-18167, No. 59-77356, and No. 59
-170768 Yongyo Official Gazette].

However, these methods have incomplete B/F separation.
There are disadvantages such as a lot of noise, problems with signal reliability, and measurable substances are limited to low-molecular substances.

[Problem that the invention seeks to solve]

On the other hand, in wet chemistry, an immunoassay method using a competitive method using a stationary phase has been developed (see, for example, JP-A-58-209994 and JP-A-59-202064). However, these measurement methods measure the entire enzyme activity without distinguishing between the two stationary phases, and therefore cannot obtain satisfactory results in terms of sensitivity, precision, and reproducibility due to background and noise problems.

On the other hand, in dry chemistry, an immunoassay method using a second antibody has been developed (Japanese Patent Laid-Open No. 57-8276).
(Refer to No. b and 57-82767 Health Bulletin). but,
There is room for improvement in these methods, as they are complicated to operate and require techniques for deploying them with good reproducibility. Furthermore, the invention described in Japanese Patent Application Laid-Open No. 59-34155 discloses a method using an unbound material storage sheet. However, even with this method, the above-mentioned problem occurs when trying to measure up to 1 with the reaction sheet and unbound substance storage sheet in close contact with each other, and it is cumbersome and troublesome to separate both sheets at the time of measurement. becomes an obstacle when automating.

The present invention has been made to improve the above-mentioned drawbacks of the prior art, and its purpose is to
By performing separation, relatively expensive molecules can be measured! The purpose of the present invention is to provide an analytical element that is applicable to 20,000 to 50,000 substances, is easy to operate, has excellent sensitivity, precision, and reproducibility, and is capable of quantifying specific components in fluid samples. .

[Means for solving problems]

To summarize the present invention, the present invention relates to an analytical element, which includes a substance B that can specifically bind a specific component A in a fluid sample, and a specific component different from the substance B. In an analytical element for measurement using a labeled material in which a label is bound to a substance O that can specifically bind to A, (
a) a carrier on which the substance B is immobilized, and (b)) the labeled substance that is not bound to the specific component A bound to the substance B.
A porous reaction layer is formed using a mixture containing a carrier immobilized with a substance (hereinafter abbreviated as absorption substance E) that binds specifically to the labeling site and modulates the signal caused by the label. It is characterized by having.

Fluid samples that can be used in the present invention include solutions and colloidal solutions in all forms, but preferably fluid samples of biological origin, such as blood, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, milk, and urine. , sweat, meat juice, etc., and blood and serum are particularly preferred.

A specific component A in a fluid sample that can be measured by the present invention is a substance whose presence or amount in the fluid sample can be measured, and two or more types of substances can be obtained that specifically bind to different sites of the molecule of the component. or a group of substances, examples of which include the substances or groups of substances listed in Table 1 below.

Table 1 (Protein) Albumin Prealbumin α “Acid Glycoprotein αt-Glycoprotein Tryptophan Deficiency α1-Riboprotein α Doglypprotein α1-Antitrypsin α2-
Glycoprotein α-Snow-ribobrotiin β-
Riboprotein β-glycoprotein C-
Reactive protein Fibrin detachment product Fibrinogen Immunoglobulin Immunoglobulin D Immunoglobulin E Immunoglobulin +
)7G Immunoglobulin M Haptoglobin Hemoglobin Ceruloplasmin
Cholinesterase hemopekikun
mycoglobin rheumatoid factor
Thyroquine-binding globulin transferrin
transcortin plasminogen
Specific antibody aggregation factors Complementary factors (peptide hormones) Adrenocorticothrobin Metho- and Leuniic 7 (MU0TH) Printelokin 7 Triiodothyronine Chorionic gonadotropin Chorionic telotropin Lucagon Inculin Neurogenesis Factors Barathyloid Hormones Placental Lactogens Probucuti/Proincniline Rexin (Tissue Hormone/) Secretin Gastrin Angiote Yryyl and U Human Placental Lactogen (Peptide Hormone from the Posterior Pituitary) Oxytocin
Vasopressin-releasing factor (R'-OR?, 'x, xp, TR7, matotrobin-R1,
GHF, P8H-RF%R engineering IIF, M engineering (cancer cell marker) Cardioembryonic Gangliosize antigen α-fetoprotein Basic 7 ditoprotein Pancreatic cancer fetal antigen Pregnancy specific β! Glycoprotein TPA Ferritin β-microglobulin Bone marrow protein astroprotein Prostate antigen Squamous cell carcinoma-related antigen 0A19-9 (microbial surface marker) Bacterial antigen Fungal antigen Parasite antigen
Viral antigen Substance B that can specifically bind to a specific component in a fluid sample that can be used in the present invention includes antibodies, antigens, lectins, protein A, inhibitors of specific enzymes, etc. However, it is particularly preferable that the binding reaction between the specific component and the binding substance is an antigen-antibody reaction. The origin of the antibodies used in the present invention is not particularly limited. Antiserum and ascites fluid obtained by administering and immunizing a supplemented animal with an antigen can be used directly, or by conventionally known methods. Oru (edited by Shunsuke Migita, “Immunity Chemistry”)
Nakayama Shoten, pp. 74-88], sodium sulfate precipitation, ammonium sulfate precipitation, Sephadex gel filtration, ion-exchange cellulose chromatography, electrophoresis, and the like.

Alternatively, it is particularly preferable to obtain a hybridoma from spleen cells of a sentinel mammal (for example, a mouse) sensitized with an antigen and myeloma cells and use a monoclonal antibody.

In addition, these antibodies are engineered for engineering gG1 engineering gM, engineering gm, engineering gD.
, and gB fractions can be used, or these antibodies can be treated with enzymes to form Fab or Fab' active antibody fragments for use. Furthermore, these antibodies may be used alone or in combination.

When an antibody or an antigen is used as the substance B that can specifically bind to a specific component in a fluid sample, the measurement principle of the analytical element of the present invention belongs to immunoassay.

Although the analytical element of the present invention can be particularly preferably used in immunoassays, it is clear from what has been described above that the present invention is not limited to immunoassays and can be applied in a variety of ways. .

Labels that can be applied to the present invention include, for example, enzymes, enzyme substrates, substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, coenzymes, prosthetic groups, substances that activate enzyme precursors, etc.). , enzyme precursors, apoenzymes, fluorescent substances, etc. Typical examples thereof include the circle substances shown in Table 2 below.More preferably, the enzymes and fluorescent substances disclosed in Table 2 are used. It will be done.

(The signals caused by these labels will be described later.) Table 2 1, Enzyme 10 1.1.1.1, Alcohol dehydrogenase 1.1.1.6, Glycerol dehydrogenase 1.
1.1.27, Lactate dehydrogenase 1.1.
1.37, malate dehydrogenase 1.1.1.4
9. Glucose-6-phos7ate dehydrogenase 5C1,1,6,4, Glucose oxidase 1.1.
3.9, Galactose Ochykdase 1.2.1.12
, glyceraldehyde-3-phos7ate dehydrogenase 1.2.3.2, xanthine oxidase 7ase 1.4.3.2, L-amino acid oxidase 1.4.
3.3, D-amino acid oxidase 1.6.4.3,
dihydroriboamide reductase (NAD)
(Diaphorase) 1.7.3.3, uric acid oxidase 1.11.1.6, catalase 1.11.1.7, beluoxidase 2.7.1.1
, hexokinase 2.7.1.2, glucokinase 2.7.1.15, ribokinase 2.7.1.28, triokinase 2.7.1.40, pyruvate kinase 2.7.5.
1, Phosphoglucomutase 3.1.1.7, Cholinesterase Bc3.1.1.8, Punidocholinesterase 3.1.5.1, Alkaline phosphatase 3.1.6.2, Acid phosphatase 3.1.3 .9, Glucose-6-phosphatase 3
．． 1.3.11, Fructo-streak phosphatase 3.
1.4.1, Phosphogesterase 3.1.4.3,
Phospholipase C 3,2,1,1, α-amylase 3.2.1.2, β-amylase 3.2.1.4, Cellulase 3.2.1.17, Muramidase 3.2.1. 18. Neuraminidase 3.2.1.2
1, β-glycosidase 3.2.1.23, β-galactocdase 3.2.1.31, β-glucuronidase 3.2.1.65, hyaluronidase 3.2.
2.5, DPHase 4.1.2.15, Aldolase 4.2, ? ,? , carbonine quanhydrase5.
5.1.1 Triose phosphate imerase zo
6.3.4.14, Shirah Zero in Biotin Carbo,
4.1.1, Hirupic acid carboxylase 6.4.1.
2. Acetyl-0oA carboxylase 6.4.1,
Propionyl-Co mucarboxylase, etc.2 Substrate p-nitrophenyl-β-D-ga5pl-cyto0-nitrophenyl-β-D-i5toside 4-methylumbelliferone-β-D-galacto7do p-nitro Phenylphosphate cortisol-21-hemisuccinate umbelliferone conjugate luminol isoluminol 1l-(4-aminobutyl)-14-ethyl hemisuccinate 6-amizhekiflu)-N-ethyl isoluminol N-( a-aminobutyl)-M-ethyl isolminol lucigenin acridinium phenyl carboxylate trophin pyrogallol gallic acid chloroquine bis(2,4,6-)lichlorophenyl]oxalate and its derivatives 5 Enzyme inhibitor physostigmine Methionine Sulfoky7mine Wildfire (Wil, dfirg) Toxic Blue Dextran 0-Dianginsin-Cellulose Q-Cyaniacin-Dextran 2-Propynylamine 2-Chloroallylamine Phenylglycine p-Nitrophenylgly7in Aminoacetonitrile 2-Amino-3- Hydroxy-lobil-1,5'-carbo-3'-amino-1'-7' lobenyl-1 ether L-2-amino-4-methoxy-trans-3-butenoic acid ethanolamine 0-sulfate albidiin aza Serine diazooxonorleucine diazoquinonoanonorbari/Δ1-7-amitusephalosboric acid mimosine 2-amino-4-pentenoic acid 2-amino-4-chloro-4-pentenoic acid 3-dichloroalanine 8 to 3 -Trichloroalanine D-Cycloserine 2-hydroxyl-5-butyric acid M, M-)Samethyl-2-propynylamine β-aminopropionitrile 2-bromoethylamine 3-decinoyl-N-acetylcysteamine 2.6-decajenoyl-N -acetylcysteamine β-chloro-
L-alani/L-serine-〇-sulfate β-fluoroalanine L-vinylglycine D-vinylglycine propargylglycungapacrine 5-nitro-L-norvaline N-benzyl-N-methyl-2-propynylamine 3-
Dimethylamino-1-propyne glycerol diisobrobyl phosphorofluoroside phenylmethanesulfonyl fluorite clapranic acid allopurinol 4, coenzyme/prosthetic group FAD (7 lapin adenine dinucleotedono FMN
(Flavin mononucleotide) Heme S-adenoflumethionine TBF (tetrahydrofolate) TPP (thiamine niline #1t) 00A (coenzyme A) UDP-GIC (uridine diphosphate glucose) pLp
(Pyridoxal phosphate) ATP (adenosine triphosphate) Biote/col to cotinamide adenine dinucleotide °]co
li cotinamide adenine dinucleotide 01 J acid] adenosylcobalamin methylcobalamin CoM (2,2'-' ditheodiethane sulfone iron) 00
Q (Ubiquinone) 5 Apo enzyme apoglutathione reductase apocytochrome reduction sarcocne ox7dase avo p-hydroxy7benzoic acid hydroxylase apoacyl-00 mudehydrogenase apodihyde a riboate dehydrogenase apocohaku l#i dehydrogenase avohomocysteine methyltransferase avoglutamate formyltransferase apotransketolase apocorinase tertransferase apoglycogen synthase avoalanine aminotrans 7 errorase avohexoginase & substance that activates enzyme precursors enteropebtedase streptokinase protein kinase various enzyme precursors grotease 7, enzyme precursor trypsinogen chymotrypkunogen / procolipase prophospholipase prorenin procarbo dipeptidase A boucarboxyk peptidase B medium collagen proelastase angiotensinogen proinsulin proparathyroid hormone proglucagon procollagen (soluble) aggregation factor XI, XI [, xm procollagenase prococonase Brekaliklein Pepsinogen Plasminogen Fibrinogen / Prothrombin Blasminogen Proactivator Gloaclodin a Fluorescent Substance Fluorescein Intiocyanate) (F Engineering To) Tetramethylrhodamine Intiocyanate (TR Engineering To)
) Rhodamine B Inocyanate (RB Engineering To) Lissamine Rhodamine-B200 Sulfolyl Chloride (R
B200SC) umbelliferone 4-'f lumbelliferone (4MU) fluorescein thiofulbamil (F T L: fluorescein thiocarbamyl-diphenylglycine (FTO-DPG) tetramethylrhodamine (TMR,) 5-((4, 6-cyclotriazi/-2-yrun-amino]fluorescein dimethylamino7talene-5-sulfonyl chloride (DNS-Ol) Fluoram 2-methoxy-2,4-diphenyl-5(2Ii)-furanone (MDPF) 7-chloro-4- Nitropenzo-2-oxa-1,3-
Diazole (NBD-07,) 1-anilino-8-naphthalenesulfonic acid (ANS) N
-(3-pyrene)-maleimide (NPM) N-(7-dimethylamino-4-methyl-2-ox7-3-chloromethyl)-maleimide (DAOM) 12-(p-2-penzimitasoylphenyl fumaleimide (B Engineering PM) Anthracene thiocyanate fluoroantylmaleimide (FAM) Various chelates containing rare earth elements and their derivatives In the present invention, the labeled material refers to a substance capable of specifically binding to the above-mentioned specific component A. This is a general term for substances to which the above-mentioned label is bonded directly or indirectly by chemical means etc. while retaining the ability to emit a signal.The above-mentioned label can be chemically bonded by various known methods, and For details, see Eiji Ishikawa, Tadashi Hedai, Kiyoshi Miyai (eds.) "Enzyme immunoassay (2nd edition)" (Igaku Shoin, published in 1978), Japanese Clinical Pathology Golden Line "Clinical Pathology" Extra Special Issue No. 53 "Clinical You can refer to the methods described in "Immunoassay for Testing - Techniques and Applications" (Clinical Pathological Publishing Society, 1983). Among these methods, the glutaraldehyde method, the periodic acid method (Nakane method) ], the maleimide method is particularly preferably used.

In addition, the "substance OJ that can specifically bind to specific component A" used for "label D" is of a different type from "substance B that can specifically bind to specific component A" immobilized on the carrier. ,
Both need to be able to simultaneously bind to different sites on one molecule of the specific component.

The absorbing substance (2) used in the present invention, that is, the substance (B) that specifically binds to the labeled site of the labeled material and eliminates or reduces the signal caused by the label, is selected depending on the label used. The following substances can be cited as examples.

1. When the label is an "enzyme," a signal caused by the enzyme activity causes a decrease in the substrate, an increase in the product, the emission of energy, and the changes caused by these. 0 Preferred absorbing substance E: An inhibitor against the enzyme (Table 1) You can select and use the inhibitor that corresponds to the enzyme from the inhibitors listed in 2.) An antibody against the enzyme that binds to the enzyme and affects its activity Z. If the label is an "enzyme substrate," 0 standard is used. Signal caused by iron Increase in products caused by the reaction of the substrate with the enzyme added to the analytical element, emission of energy, and changes caused by them 0 Preferred absorption substance E An antibody against the substrate that binds to the substrate A substance that inhibits the enzymatic reaction by doing so.

An enzyme that takes up the substrate as an irreversible inhibitor An enzyme that uses the substrate as a substrate, and is originally intended to be detected by its reaction (one that does not emit No. 8) ”, then 0 decrease in substrate due to the reaction of the enzyme that requires the label added to the signal analysis element due to the label, increase in products, and changes caused by them 0 Preferred absorption substance Z for the label An antibody that binds to the label and affects its activity.An antibody that absorbs or consumes the label, but does not emit the signal that is originally intended to be detected due to its activity.

4. When the label is “apoenzyme” Signal caused by zero pointer The label does not emit a signal as it is.

It binds to the absorption substance described below to express enzymatic activity, and it is possible to measure the decrease in substrate and increase in product due to this activity, as well as the changes caused by these.

0 Preferred absorption substance 2 A prosthetic group that expresses the enzymatic activity of the label. (You can select and use the one corresponding to the label from the prosthetic groups listed in Table 2.) If the label is a "substance that activates the enzyme precursor," the signal caused by the ovA shoe is detected in the analytical element. Activates the enzyme precursor added to the enzyme, and its activity causes a decrease in the substrate, an increase in the product, and changes caused by these.0 Preferred absorption substance E: An antibody against the substance, which binds to the substance and affects its activity. If the substance is an enzyme, the inhibitor & label is an "enzyme precursor". 0 Signal caused by the label. The label does not emit a signal in 11.

Once bound to the absorption substance described below, part of the molecule is cleaved and enzyme viscosity is developed, and the decrease in substrate and increase in product due to its activity and changes caused by these can be measured.

O Preferred absorbing substance - Substance that expresses a certain enzyme activity If the label is a "fluorescent substance" - Signal caused by the label - Fluorescence emitted when the fluorescent substance is irradiated with excitation light - Preferred absorbing substance E - Antibodies and derivatives thereof against labels that change the fluorescence wavelength and intensity of the label.

Specific examples of the various absorbent materials described above are all well known to those skilled in the art and need not be disclosed here, but representative examples are shown below to aid understanding of the present invention.

Combinations of enzymes and inhibitors that can be used in the present invention include biotene enzymes (pyruvate carboxylase, acetyl
0-mucarboxylase, propionyl-00 mucarboxylase, methylmalonyl-COA carboxylase, etc.) and avidin, peroxidase and Q-cyanidin-dextran, lactate oxidase and 2-hydroxyclue-5-butic acid monoamine or? 7dase and N.

Examples include N-trimethyl-2-propynylamine or β-aminopropionitrile.
Am, Ohem, soa) Volume 80, Page 456 (B958); Volume 82, Page 596 (B960); Accountant Chemical Research (Ace,
Chem, Res) Volume 9, page 513 (b976); Science (8cie-nce) Volume 185, 32
0 members (b974); Kagaku Kogyo 1985, page 21 (b9
Combinations of enzyme inhibitors described in 1985) or others cited can also be preferably used.

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

The particles used here preferably have a particle size of 1 to 1000 μm, and the materials include dextran, agarose, polyamide, polystyrene, polycarbonate, organic polymer particles such as those disclosed in JP-A No. 55-90859, or Inorganic particles such as glass, silicon dioxide, and sand are preferably used, but it is particularly preferable to use self-bonding particles disclosed in JP-A-57-101760 and JP-A-57-101761. Typical materials include those shown in Table 3 below.

Table 5 Exemplary compound poly(styrene-coglycidyl methacrylate)
C90/10] Poly(sterene-co-methyl acrylate)
Poly(styrene-two-n-butyl methacrylate-co-glycidyl methacrylate) [80/1515] [75/15/fO co.

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

Poly(styrene-codivinylbenzene-co-glycidyl acrylate) C90/2/8].

(C6 poly(p-vinyltoluene/-cochrycidyl methacrylate) [90/10].

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

Poly(styrene-N, N-dimethylamine ethyl methacrylate) C9515.

Poly(styrene-coaziridyl ether methacrylic sheet) C9515].

αq poly(styrene-comederacrylate-coacrolein) Cq o1515 ].

Poly(styrene-co-acrylamide) C9515] Poly(styrene-co-vinyl theol) C9s153゜αJ Poly(styrene-co-methylolated acrylamide)
C9515].

α◆ Poly(styrene-coat t-7'f acrylate-glycidyl methacrylate)) C951515].

(G) Poly(styrene-covinyl incyane-)) [ep
515].

αQ poly(methelacrylate-costerene-co-N-methylolacrylamide) [50/S 5/15].

Poly(sterene-co-glycidyl methacrylate)
111,N-dimethylaminoethyl methacrylate -
) (: 9 01515
].

Poly(styrene-co-methacrylic acid-co-acrylamide) (95/2/5 pieces.

(To) Poly(styrene-N-methylolacrylamide-co-acrylic acid methoxyethyl tv) [951515].

(e), l-' IJ (p-hinyltoluene-co N-/
Tyrol acrylamide co-acrylic acid) [90/8
/2].

Qυ Poly(methyl methacrylate-co-gly7yl methacrylate-co-t-butyl acrylate)) C8Q
/1 0/1 0].

Poly(sterene-co-p-vinylpe/silchloride-co-acrylic acid-co-ureidoethyl acrylate) (
75/1015/10].

Kanpoly (styrene-co-metacrolein-co-α-hydroxyethyl methacrylate) [?01515].

(c) Poly(styrene-coacrolein-coacetoacetate-IP7 ether methacrylate) C8515/10)
.

Poly(styrene, N-dimethylaminoethyl acrylate, vinylsulfonyl ethyl methacrylate) [9015153°) yN! J (p-vinyltoluene-co7mi/styrene-covinylsulfonyl ethyl methacrylate), 1c
85/1015].

(b) Poly(styrene-N, N-dimethylaminoethyl methacrylate) Cqo/1o].

(To) Poly(Suderene-coacrylic acid) C97/3].

■ Poly(suderene-co-acrylamide) [97/3] Soho U (p-hinyltoluene-co-t-p-f acrylate) C9515] Poly(methyl acrylate-co-methacrylamide)
C9515] 0'4 Poly(styrene-to-N-methylolacrylamide)
[9s15] (to) Poly(p-vinylbenzylchloride-co-N-methylolacrylamide) C96/a ] (b) Poly(styrene-coitaconic acid) [:98/2] (g) Poly(styrene-two-t) -butyl acrylate))
C92/8] (to) Poly(methyl acrylate-co-styrene-co-acrolein) [50/6 s15] (b) Poly(methyl methacrylate-co-styrene-co-2-
Hydroxyethyl methacrylate) C2s/yo
/ 53 Competitive poly(sterene-co-vinylsulfonyl ethyl acrylate)) C80/20 ] (To) Poly(sterene-co-N,N,-dimethylaminoethyl acrylate)) [90/101] yNIJ (Xf Ren-methyl Acrylic acid-covecetoacetoxyethyl acrylate) [901515] 1υ Poly(styrene-comethacrylic acid) [95-15] The weight of the monomer used in the polymerization reaction is shown in parentheses after each exemplified compound. .

Alternatively, several kinds of these O particles can be used in combination.

In addition, examples of fibers used in the porous reaction layer of the present invention include pulp, cotton, linen, silk, wool, cellulose ester, viscose rayon, cuprammonium rayon, polyamide (6
-nylon, 6-nylon, 410-nylon, etc.)
, polyester (such as polyethylene terephthalate),
Polyolefins (polypropylene, vinylon, etc.), glass fiber, asbestos, etc. Plant-based, animal-based, mineral-based, synthetic,
Semi-synthetic/regenerated fibers may be used, and lupine may also be used in combination.

The substance that specifically binds to the specific component can be immobilized by physically adsorbing the substance to the surface of the particles or fibers, or by directly or indirectly bonding the substance to the surface of the particles or fibers through a chemical reaction, using various known methods. Especially achieved. At this time, it is necessary to take care not to lose the specific binding property of the substance to the specific component.
1978), “Experiments and Applications Affinity Chromatography” by Kazu Chibata, Tetsuya Tosa, and Yushi Matsuo (Kodansha 1)
976] can be cited as an example of a preferred method.

In addition, after immobilizing the substance B that specifically binds to the specific component A, it is possible to carry a protein that is not involved in the immune reaction to be measured, if necessary, in order to eliminate non-specific reactions in the immune reaction. It's possible.

Typical examples of these include normal serum protein, albumin, gelatin, and its decomposition products from dairy animals.
Ru.

Having said the above, create (a) and (b)) using the method /r'! Ru. In this case, (a) and ('b) do not necessarily have to be made of the same material; particles of different filter materials, fibers of different materials, or a combination of particles and fibers may be used. . However, when L2, (n,) and (b1) are uniformly combined as much as 6 'f2-me VC, ore, the average grain size is significantly 1-<75:
lli particle? Combination is not recommended.

The mixing ratio of particles or fibers (a) and ('b) is determined according to the binding constant (C) of each specific binding substance immobilized on each, and if necessary, the specific binding substance is Immobilization I
, particles other than 5 or Fy, fibers may be added for adjustment.

By applying and/or not forming a film with these mixtures,
Porous pores with interconnecting pores that come into free contact with the fluid sample 1,7+! A porous reaction layer having a t' structure is formed. Particles that do not have self-bonding properties can be formed into a film by point-adhering the particles to each other using a suitable adhesive, for example, the method disclosed in JP-A-55-9O859 can be applied.

2) Organic polymer particles having self-bonding properties are disclosed in Japanese Patent Application Laid-open No. 1983-
No. 1017 or No. 57-t01761 Δ Films can be similarly formed by the methods described in each publication.

A porous reaction hole can be formed by applying the fiber dispersion described in JP-A-57-125 [347 and JP-A-57-197466] to a fiber or a T fiber and a particle mixture. , trench patent application 11 (359-28571
It is also possible to apply a fiber dispersion using a water-based binder such as gelatin or polyvinylpyrrolidone, as in the method described in No. 1.

The shape of the analytical cable t of the present invention is not particularly limited as long as it can be used for analysis, but from the viewpoint of manufacturing, operation, and evaluation, it is preferably in the form of a film or a foot.

7C, which helps the analytical element of the present invention, -1
The principle will be explained using flJ.

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

FIG. 2 is an enlarged view of FIG. 1. In Figure 2, number 2
is the particle of (a) above, 5 (fl) is the particle of (b) above, and 4 is the particle added for adjustment.

FIG. 2 shows that particles 2.3 and 4 are evenly mixed and bonded together to form a porous reaction layer.

FIG. 3 is a schematic diagram of a label, where 8 is a substance O59 that can specifically bind to a specific component A in a fluid sample;
10 means a sign.

FIG. 4 is a schematic diagram illustrating the present invention, in which reference numeral 5 is a substance B (different from substance C) that can specifically bind to a specific component A in a fluid sample, 6 is an absorbing substance m, and 7 is a fluid. It means specific component A in the sample.

1 to 4, the mechanism of the present invention will be explained.

Drop a certain amount of fluid sample f) onto the analysis element shown in Figure 1 using a scale.3 Then, the specific component A in the fluid sample is immobilized on the surface of the particles or fibers. substance B that specifically binds to specific component A.

Next, when liquid m containing the labeled material is dropped, the labeled material 111
:, specific component A adsorbed on the particle surface of (a) and (b)
)) is distributed between the absorbent substances Z immobilized on the particle surface (Fig. 4).

For a certain reaction time, the labeled substance is adsorbed via the substance B onto the particle surface on which the substance BJ, which specifically binds to a specific component in the fluid sample, is immobilized.

The absorbent substance is immobilized and adsorbed to the immobilized surface through the substance.

C. There are 03 states remaining in the fluid sample.13. It is preferable to set the reaction conditions and the structure of the porous reaction layer so that the proportion F'i of these is as small as possible. Specific component A in the fluid sample (
The proportion of A is controlled by the concentration of b0, and the higher the concentration of the specific component A, the higher the proportion of G' becomes, and the lower the proportion of G'. In the case of a marker in the oral state, the signal caused by the marker is modified by the combination of the marker site and the absorbing substance, so the concentration of the specific component A in the fluid sample and the overall image of the marker are changed. A functional relationship is established between the No. 1 intensities. Therefore, if you create a calibration curve using a fluid sample (standard sample) in which the concentration of specific component A is known, you can find out the concentration of specific component AO in an unknown fluid sample. It becomes like this.

The method of measuring the signal varies depending on the type of sign.

For example, if the label is a fluorescent substance, excitation light may be applied to the analytical element and the fluorescence intensity may be measured. If the label is an enzyme, add a suitable substrate, if necessary, a solution containing an enzyme or a coloring system, and incubate for a certain period of time. The signal strength can be measured by measuring the luminescence intensity). Substrates and coloring systems used for this purpose are labeled a#.
An appropriate method can be selected from known methods according to the type of element.

For example, when the label is peroxidase, a buffer (pH
40 to 9.0), a solution of one or more of the compounds exemplified in Table 4 can be used.

Table 4 (b) 0-cyanidine (2) o-tolidine or its acid salt (3) 0-phenyl diamine or its acid salt (4)
Guaiac (phrase adrenaline (6J phenol 7-thaley/ (7) ferrocyanide (8) combination of 4-aminoantipyrine and its derivatives or their acid salts with phenol or naphthol or their derivatives (9) anili/ and Derivatives thereof (b10-) Monoamines such as luidine and P-toluidine aη Q-phenylenediamine, N,N'-dimethyl-
Diamines such as p-7 22 diamine, \x'-diethylphenylene diamine, benzidine, dianisidine, etc. (b) Phenol, thymol, 0-, ・m- and hip-
Phenols αJ such as cresol, α-naphthol, β-naphthol, catechol, guayol, orcinol, pyrogallol, easy p-dihydroxydi7-dyl, 70 log)
Polyphenols such as vS/Nol Aromatic acids such as salicylic acid, pyrocatechinic acid, gallic acid - Leuco dyes such as leucomalachite green, leucophenolphthalein α11 2.6-') chlorophenolindophenol Various biochemical substances such as epinephrine, flavones, dihydroquine, phenylalanine, tryptophan, etc. 2. or its salts, and special dyes such as 3'-diaminobenzidine, as well as substance labels such as guaya gum, guaiaconic acid, potassium iodide, sodium iodide and other water-soluble iodides, and bilirubin. The same is true for substrates, coenzymes, apoenzymes, substances that activate enzyme precursors, and enzyme precursors: 1!1υ, in short, add a solution of the substance required for signal measurement, incubate, and measure the reflection concentration. , fluorescence intensity, luminescence intensity, etc. may be measured.

Microscopically, the particles or fibers of the carrier shown in (IL) and (b)) used in the present invention constitute a reaction layer with their surfaces separated except for a slight adhesive part. This secures a space for accommodating the fluid sample, and at the same time, the labeled part of the labeled material adsorbed to the substance B immobilized on the surface of the particle or fiber of (a) is the particle of (b) or This prevents the signal from being modulated by the effects of substances fixed on the surface of the fiber. As a result, B/F separation within a single layer, which is a feature of the present invention, became possible.

Furthermore, another advantage of the present invention is that when mixing the particles or fibers (&), (b)) and the particles or fibers added for adjustment if necessary, the ratio can be set freely. , (a) and (b) K: Even if the amount of immobilized substances and the ability to bind to labeled materials vary somewhat from manufacturing loft to manufacturing loft, the performance always remains constant by adjusting the mixing ratio. It is possible to supply elements. Conventional analytical elements and similar products that utilize a porous layer made of particle or fiber aggregates treat particle or fiber aggregates simply as containers for storing fluid samples; The attempt to perform B/F separation in a single layer by mixing and forming films with different functions is a completely new attempt, and the fact that the above-mentioned effects were actually obtained is a great achievement for the inventor. This was a completely unexpected and surprising result.

Various embodiments are possible in implementing the invention.

For example, when using the analytical element of the embodiment shown in FIG. 1, in addition to the method described above, a fixed amount of the labeled substance and the fluid sample may be mixed in advance and then dropped, or the fluid sample and the labeled substance may be mixed together. It is also possible to drop the solutions simultaneously. Although this method has some disadvantages in sensitivity, on the other hand, it can greatly simplify the operations for analysis.

Although the above-mentioned porous reaction layer is the minimum necessary component of the analytical element of the present invention, various auxiliary layers can be provided to further exhibit the effect of the reaction. FIG. 5 to FIG. 10 are schematic cross-sectional views showing one embodiment of the analytical element of the present invention.

The analytical element of the present invention shown in FIG.
A porous reaction layer is laminated on top of the support, and the presence of the support improves the handling of the device.

Supports that can be used for this purpose include polymeric compounds such as cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds (eg, polystyrene), or transparent inorganic compounds such as glass. The porous reaction layer may be coated and/or hardened directly on such a support, or the porous reaction layer may be formed separately and then applied to the support. In the embodiment shown in FIG. 5, the fluid sample must be dropped from the reaction layer side, but signals can be measured from both sides.

In another embodiment of the invention shown in FIG. 6, a reactive layer is provided on the light reflective support 12.

In this embodiment, both sample dropping and signal measurement are performed from the porous reaction layer side, and the light-reflecting support facilitates the measurement of signals by reflection density (when measuring signals by fluorescence intensity, A similar effect can be achieved by using a black light-absorbing support).

Support materials that can be used for this purpose (7) In addition to the above-mentioned support materials, examples include ceramics, metals, or paper coated with resin to make it waterproof. If the material is desired, the purpose can be achieved by coating or containing a white pigment such as T10!, B a S04, mica, etc.

The embodiment shown in FIG. 7 has a similar purpose, and a porous reaction layer 1 and a light reflecting layer 13 are laminated in this order on a light-transmitting support 11. In this embodiment, the sample is dropped from the light-reflecting layer side, and the signal measurement is performed from the light-transmitting support side. The light-reflecting layer can 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 coated with the aforementioned white pigment.
#+ etc? It is possible to apply, form a film, or paste the containing material. More preferably, substance B or absorption substance 11ff, which specifically binds to a specific component in a fluid sample, is immobilized on the surface of particles containing a white pigment inside, as in the porous reaction layer. A light reflecting layer having the same function as the reaction layer can be provided.

By using such a special light-reflecting layer, it is possible to prevent a large amount of unreacted label from remaining in the light-reflecting layer.

In the embodiment shown in FIG.
4 are provided. The labeled substance-containing layer is a layer in which a labeled substance is contained in a porous medium so that the area concentration is constant,
When a certain amount of the fluid sample is dropped, a certain amount of the label is eluted and diffused into the porous reaction layer along with the sample. As for the material, the same material as the porous reaction layer may be applied, formed into a film, or pasted, or water-absorbent Western paper, Japanese paper, door paper, plush polymer, glass fiber, mineral fiber (
asbestos, etc.), vegetable fibers (cotton, hemp, bulbs, etc.), animal fibers (wool, silk, etc.), synthetic fibers (various nylons,
Vinylon, polyethylene terephthalate, polypropylene! ``J, A label-containing layer made of woven fabric, non-woven fabric, synthetic paper, etc. made of rayon, cellulose ester, etc. alone or in combination may be attached. This label-containing layer can also have the effect of the light-reflecting layer described above.In the embodiment shown in Figure 8, if the label is a fluorescent material, measurement can be performed immediately by dropping only the fluid sample and incubating it. When the label is another substance, measurement can be performed by dropping a fluid sample, incubating it for a certain period of time, and then dropping a solution containing the necessary substrate, coloring reagent, etc.

FIG. 9 is another embodiment having a label-containing layer. In this case, from the bottom, the light-transmitting support 11, the label-containing layer 14
, a timing layer 15, and a porous reaction layer 1 are laminated. The timing layer is a 6D layer, for example, a 9 gelatin layer with appropriately adjusted hardness, which is a technique widely known in the field of photochemistry. In this embodiment, after the fluid sample is dropped, the specific component in the sample is adsorbed to some extent on the surface of the particles in the porous reaction layer, and then the marker is released into the porous reaction layer. Such a method is widely known as delayed addition, and is particularly effective in the present invention.

When the label-containing layer is located in a portion other than the top layer as in this embodiment, materials used for the label-containing layer include gelatin, gelatin derivatives, polysaccharides (agarose, etc.), carbogial methyl cellulose, hydrocarbons, gelatin derivatives, polysaccharides (agarose, etc.), Hydrophilic polymer substances such as diethyl cellulose, vinyl pyrrolidone, acrylic acid conductor, methacrylate/I/@ derivatives, vinyl alcohol, sulfonylstyrene, etc. are used as monomers, and a continuous binder such as a homopolymer or copolymer is applied. Another method is to control the extrusion rate of the label from the label-containing layer by adjusting the film thickness and composition of the hydrophilic polymer or polymer, thereby improving the function of the timing layer. It is possible to make it also serve as.

The embodiment shown in Figure i1o is particularly useful when the label is other than a fluorescent substance (in the case of a label that utilizes some form of enzymatic reaction for signal measurement).

The reagent layer 16 is made of the same material as the marker 14, and contains a substance necessary for measuring a marker signal at a constant area concentration. By providing the reagent layer and the label-containing layer, even if a label other than a fluorescent substance is used, measurement can be performed by simply dropping a fluid sample and incubating it.

In this case, it is preferable that the contents of the reagent layer are eluted after the reaction in the porous reaction layer has sufficiently progressed, and for this purpose, the above-mentioned timing layer may be provided on the reagent layer, or the composition of the reagent layer may be changed. It is necessary to adjust it to have the effect of a timing layer. For the same reason, it is preferable to provide the reagent layer on the bottom layer of the analytical element (or above it if a support is included).

In addition, 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 difficult to contain directly into the device, so cumene/H! Preferably, hydrogen peroxide is contained in the form of hydrogen peroxide, such as 02, or generated at the time the fluid sample is dropped. The latter uses, for example, oxidizing enzymes (types that produce hydrogen peroxide as a reaction product) such as glucose oxidase, urate oxidase, and amine oxidase, and the substrate of the enzyme in a reagent layer in a dry state. Alternatively, one of the oxidase and the substrate may be contained in a reagent layer, and one r-+: male may be contained in a different layer.

FIGS. 11 and 12 show a cross-sectional view and a perspective view of an example of a preferred embodiment of the present invention. In order to facilitate the handling of the analytical element, the entire body is covered with a mount 17 made of Pubstick 29, and a sample injection hole on the top of the mount,
There is a signal Δ measurement hole at the bottom.

The analytical element of the present invention further includes a development layer that assists in the development of a fluid sample when it is applied to the element;
A blood cell separation layer, an adhesive layer, a protective layer, and an auxiliary layer may be provided as needed. These auxiliary layers, the above-mentioned coloring reagent layer, label-containing layer, and timing layer may be provided independently, or may be provided as a layer having multiple functions. The positions where these layers should be provided can be easily determined depending on their functions.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to examples, but the invention is not limited to these examples.

Example 1 (b) Preparation of rabbit anti-FTC antibody 50q of bovine serum albumin (BI3A) was dissolved in 1% charcoal) I
A solution of SL and 2av of fluorescein inteonanate (manufactured by Research Organix, USA) in 500 pL of dimethylformamide was added to a solution of sodium hydroxide (pH 9,0), stirred at room temperature for 5 hours in the dark, and Sephadex G was added. -25 (7) was purified using a fucolumn manufactured by Almac 7 and lyophilized.

This was mixed with 70 India complete adjuvant (first time only) and incomplete adjuvant, and rabbits were immunized.

The obtained antiserum was separated into a globulin fraction by the ammonium silosulfate method, and B8 was analyzed by affinity chromatography.
After removing antibodies adsorbed to the membrane, the mixture was dialyzed and freeze-dried.

(2) Preparation of porous reaction layer (7) Goat anti-human egg gG (manufactured by Cupbell, USA) f:
10 μg/d in α05M pH 9,4 carbonate/bicarbonate buffer
Poly(styrene-2-n-butylmethacryl'-to'-gly△dylacry'-)) [75/1s/1ol of high molecular weight polymer particle units with an average particle size of 21 μm were added and heated at 4°C. I left it there overnight.

After washing the particles with physiological saline, they were placed in a similar buffer solution containing 200 μg/- of E8A, left at 4° C. for 3 days, and washed with physiological saline.

(b) Anti-F engineering T prepared in (b) using anti-human 1g GO substitute
.

The same operation as (7) was performed using the antibody.

(Re) Particles with only BSA physically adsorbed were created using a method similar to (7).

The particles prepared in (7) to (fA) were mixed uniformly in a ratio of 7:1:3C1, and 5 parts by weight of Triton X-100 (
The film was coated on a polyethylene terephthalate support together with a nonionic surfactant (manufactured by Rohm End Haas) to a dry film thickness of 350 μmK, dried at 42° C. for 30 minutes, and after the film was formed, it was peeled off from the support.

(3) Preparation of label-containing layer A reagent layer having the following composition was coated on a drawn polyethylene terephthalate film and dried.

(4) Preparation of analytical element The porous reaction layer prepared in (2) was adhered onto the marker-containing layer prepared in (3), and cut into a size of 2×2011 to prepare an analytical element.

(5) Measurement of human egg G A 001M sodium phosphate buffer (pH 7, 6) containing human egg G (manufactured by Cavbell, USA) at various concentrations from 640 μg/- to 0 μg/- is prepared.

10 μt of XgG solution of each concentration was dropped onto one analytical element pot prepared in (4), and after incubation at 37°C for 20 minutes, fluorescence measurement was performed from the side where the sample was dropped (excitation wavelength: 490 nm, fluorescence wavelength: 520 nm). However, we obtained the results shown in Table 5.

Table 5 Example 2 (b) Creation of reagent layer The following coloring reagent layer (mc) was coated on a subtracted polyethylene terephthalate 0 film and dried.

(2) Creating a label-containing layer (created with υ and applying the following label-containing layer on top of the drug layer of the following formula,
Created by drying.

(3) Introduction of reactive groups into cellulose fibers 55 tons of Powder 2 Paper D (manufactured by Toyo F Paper Co., Ltd.) was soaked in 1000-2.5M potassium phosphate buffer (pH 12.1) for 5 to 10 minutes.
While stirring with a stirrer at 0.degree. C., a 500-promoquane solution (b052/sg) was gradually added. 2
Distilled water, 11M, filtered and ice-cooled after 0 minutes of reaction.
The mixture was washed with hydrogen carbonate and IJum in sequence.

(4) Creation of porous reaction layer (7) Goat anti-human egg G (manufactured by Cupbell, USA) G
a fraction tOq (antibody tanned daily amount conversion) to αI of 40-
M NaHCOl -CL 5 M Hoot
A portion of the activated F paper prepared in step (3) was dissolved in the layer solution, and the mixture was shaken at room temperature for 3 hours. - Retire, shake with 1M Tris-HCl buffer (pHaO) for 2 hours at room temperature.

Water, (b5M acetate buffer (pH 4,0), 15M Na
HCOl, 50 mM phosphate buffered saline (p
Wash sequentially with H7, 2). This was immersed overnight in 50mM Tris/HCl buffer (pHaO) containing two major B8 molecules, washed with distilled water, added with a small amount of an aqueous solution of B8A and sucrose, and then lyophilized.

(a) A portion of the activated p-paper prepared in (3) was reacted with 0-dianisidine at pH 9 overnight at room temperature in the dark. -Retirement 1
M After treatment with 1-amino-2-propatool and washing with water (
BsA treated and dried in the same manner as in 7).

(fj) The same operation as in (7) was performed using glucose oxidase and a part of the activated 2 paper prepared in (3).

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

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

(Oki of human egg G) A 1M sodium phosphate buffer solution (pH 7.6) containing constant human egg G (manufactured by Kappel, USA) at a concentration of 640 μg/- to 0 μg/ad is prepared.

10 μL of human IgG solution at each concentration was dropped onto each analytical element prepared in (4), and after incubation at 37° C. for 20 minutes, the reflection density at 492 nm was measured from the support side. The results are shown in Table 6. 6 [Effects of the Invention] As explained above, according to the analytical element of the present invention, B/F separation is performed within a single layer, and the sensitivity and accuracy can be improved by simple operation. It is also possible to achieve the remarkable effect of being able to quantitatively analyze a specific component having a relatively high molecular weight in a fluid sample with good reproducibility.

[Brief explanation of drawings]

Figure gA1 and Figures 5 to 10 show one of the analytical elements of the present invention.
2 is an enlarged view of FIG. 1, FIG. 5 is a schematic diagram of a sign, FIG. 4 is a schematic diagram illustrating the present invention, and FIG. 11 is a preferred embodiment of the present invention. A cross-sectional view of one example of the embodiment, and FIG. 12 is a perspective view of FIG. 11. 1: porous reaction layer, 2: carrier (a), 3: carrier (b))
, 4: Particles added for adjustment, 5: Substance B. 6: Absorbing substance E, 7: Specific component A in the fluid sample.

Claims (1)

[Claims] 1. A substance B that can specifically bind a specific component A in a fluid sample, and a specific component A different from the substance B.
In an analytical element for measuring using a labeled substance D, which is a substance C that can specifically bind to a label, and a label, (a)
(b) specifically binds to the carrier on which the substance B is immobilized, and (b) the label site of the label D that is not bound to the specific component A bound to the substance B, and modulates the signal caused by the label. An analytical element characterized in that it has a porous reaction layer formed using a mixture containing a carrier on which a substance E is immobilized.