JPH02263162A - Analyzing element - Google Patents

Abstract

PURPOSE:To measure the specific component in fluid with a high sensitivity and low noise by forming a reaction layer contg. hydrophilic fine particles, fine particles having a hydrophobic group and hydrophilic binder in such a manner that the ratio of the hydrophilic fine particles and the fine particles having the hydrophobic group attains a specific range. CONSTITUTION:This element has the reaction layer contg. the hhdrophilic fine particles fixed with a material A which bonds specifically to the specific component or a material B which bonds specifically to the material A, the fine particle having the hydrophobic group and the hydrophilic binder. The layer is so formed that the ratio of the hydrophilic fine particles and the fine particle having hydrophobic group is 5:1 to 60:1 by weight. The reaction layer referred to is the field where the specific bond reaction of the material fixed to the carrier and the specific component in a liquid sample or the specific bond reaction of the material fixed to the carrier and the specific component in the liquid sample rises. The specific component in the fluid is detected with the high sensitivity, low noise and good accuracy in this way.

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 immunological analytical element for measuring a specific trace component in a biological fluid sample.

[Prior Art] Various analytical methods have been developed to detect substances contained in trace amounts in biological fluid samples. The analysis method is mainly based on the immune reaction.

Although various measurement methods have been proposed using the above principle, immunoassay is known as the most accurate method.

[Disadvantage of conventional technology 1 However, one of the important technical problems regarding the above immunoassay method is that the substance that caused binding (hereinafter abbreviated as B) and the substance that did not bind (hereinafter abbreviated as F) )
separation (hereinafter abbreviated as B/F separation). Traditionally, B/F separation involves contacting an antibody immobilized on a solid phase with a fluid sample and a labeled antigen or antibody, and then repeatedly washing the same phase to remove F. As a solution to this problem, various methods have been disclosed to simplify the operation by applying immunoassay to dry chemistry, which has been attracting attention in the field of clinical biochemical testing in recent years. (For example, JP-A-53-38619
No. 53-79024, No. 5590859, No. 5
No. 7-67860, No. 57-200862, No. 58-
No. 18167, No. 59-77356 and No. 59-17
No. 0768).

However, these methods provide incomplete B/F separation. Further, there are drawbacks such as a lot of noise, problems with signal reliability, and measurable substances being limited to low-molecular substances.

As a means to solve these conventional B/F separation problems, methods have been developed in recent years that use substances that specifically bind to unreacted labeling substances and modulate signals caused by the labeling substances.

This technology is disclosed in Japanese Patent Application Laid-Open Nos. 61-292060 and 62-90.
No. 539, No. 62-90538, No. 62-24725
6, No. 62249063, No. 62-249060, No. 62-267666, No. 62-267667, No. 61-280166, etc.

However, all of these immunoassay techniques applied to dry chemistry are characterized by immobilizing a substance that undergoes a specific binding reaction on the surface of carrier particles constituting the reaction layer. Therefore, if a water-insoluble binder is used to form the reaction layer, the binder will coat the surface of the carrier particles, effectively deactivating the substance that performs the specific binding reaction at that part. This has been a major obstacle to improving analytical sensitivity.

In addition, if a water-soluble binder is used, it is possible to avoid such deactivation due to coating, but the binder dissolves in the small amount of water contained in the voids of the reaction layer, making it extremely difficult. It was discovered that the analytical reaction had to be carried out in a highly concentrated hydrophilic banhgu solution, which caused the distribution of water in the analytical element to become unrestricted over time during incubation, significantly reducing the analytical sensitivity. It was done.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide an immunological analysis element for measuring a specific component in a fluid with high sensitivity and low noise.

[Means for Solving the Problems] As a result of studying the above-mentioned techniques, the present inventors discovered that highly sensitive measurements can be performed by adding fine particles having a hydrophobic group to the reaction layer in the analytical element. Completed the invention.

That is, the present invention provides an analytical element that measures a specific component in a fluid sample by a binding reaction with a substance A that specifically binds to the specific component. It has a reaction layer containing hydrophilic fine particles on which A or a substance B that specifically binds to the substance A is immobilized, fine particles having a hydrophobic group, and a hydrophilic baingu, and has the hydrophilic fine particles and the hydrophobic group. The ratio of fine particles is 5:1 on a weight basis
60.1 is provided.

[Effects of the Invention] The present invention provides an immunological analysis element that detects a specific component in a fluid with high sensitivity, low noise, and high accuracy compared to conventional analysis elements.

[Detailed Description of the Invention] The reaction layer in the present invention refers to a specific binding reaction between a substance fixed on a carrier and a specific component in a liquid sample, or a reaction between a substance fixed on a carrier and a specific component in a liquid sample. This is the place where specific binding reactions with substances occur.

Examples of the fine particles having a hydrophobic group used in the analytical element of the present invention include vinylpyrrolidone/styrene copolymer, ethylene/vinyl acetate copolymer, acrylamide/styrene copolymer, acrylamide/isoprene copolymer, etc. . Among these, particularly preferred are:
Mention may be made of water-insoluble polymer latexes having an average particle size of 0.1 to 1.0 μm. In particular, a copolymer of a monomer having a hydrophobic group and a monomer having a hydrophilic group is preferable. The mixing ratio of the monomer having a hydrophobic group and the monomer having a hydrophilic group is 1:100 to 100:1 in molar ratio. Preferably the ratio is 1:5 to 5:1. Examples of monomers having hydrophobic groups are shown below, but the invention is not limited thereto.

(However, in the formula, R' and R2 are the same or different, a hydrogen atom, a halogen atom, a substituent such as an alkyl group or an aryl group having 1 to 10 carbon atoms that does not contain an optionally substituted amino group, R3 represents an aliphatic group or aromatic group having 1 to 10 carbon atoms and not containing a hydrogen atom, a halogen atom, or an optionally substituted amino group. As aliphatic groups and aromatic groups,
Examples include an alkyl group, an alkoxy group, an aryl group, and an aryloxy group. Examples of the monomer represented by the above formula include styrene, vinyltoluene, vinylbenzyl chloride, and butylstyrene.

(2) R CH2==C 0OR2 (However, R1 represents a hydrogen atom or a methyl group, and R2 represents a methyl group, an ethyl group, or a propyl group.) (31R'
R2 CH2=C-C=CR2 (However, R R2 is different and represents a hydrogen atom or a methyl group.

CH2=C (However, R represents a hydrogen atom or a methyl group.) (51
CH2=CH C0R (R represents a methyl group, ethyl group or propyl group) Examples of monomers having a hydrophilic group are shown below, but are not limited thereto.

(6) R' / H2C (nee H CR2=CR2 (However, R1, R2, R3 and R4 are the same or different, a hydrogen atom, a halogen atom, an optionally substituted alkyl group having 1 to 10 carbon atoms, or (Represents an aryl group.) (7) R CH2=C 0NH2 (However, R represents a hydrogen atom or a methyl group.) (8)
R CH2=C CONR2R” (However, R1 and R2 represent a hydrogen atom or a methyl group, and R3 represents a methyl group or an ethyl group.) (9) R CH2=C H (However, R represents a hydrogen atom or a methyl group. ) Examples of copolymers of the above monomers having a hydrophobic group and monomers having a hydrophilic group which can be preferably used as fine particles having a hydrophobic group in the present invention are listed below.

However, in the above copolymer (1) to 6), m
and n are in a molar ratio of 1:100 to too:1, preferably 1:5 to 5.1.

These copolymers can be produced, for example, by radical polymerization of the above monomers by suspension polymerization or emulsion polymerization using azobisisobutyronitrile, benzoyl peroxide, potassium persulfate, ammonium persulfate, etc. as an initiator. can.

In another embodiment, (7) hydroxyethyl cellulose (8) carboxymethyl cellulose alkali salt (9) a monomer having the above-mentioned hydrophobic group in a wood-loving polymer such as polyvinylpyrrolidone (
Examples include those obtained by graft polymerization of 1) to (5).

Preferred examples of the hydrophilic fine particles used in the analytical element of the present invention include microcrystalline cellulose, cellulose powder, crosslinked dextran (Sephadex (manufactured by Pharmacia),
Sephacryl (manufactured by Pharmacia), agarose,
Examples include water-insoluble polysaccharide fine particles such as cross-linked agarose (cellulose powder, etc.). Further, the average particle size of these particles is preferably 1 to 100 μm, but they do not necessarily have to be spherical and may be irregularly shaped.

Furthermore, as the water-soluble binder used in the analytical element of the present invention, monopolymers or copolymers containing vinylpyrrolidone, acrylic acid derivatives, methacrylic acid derivatives, vinyl alcohol, sulfonylstyrene, vinyl acetate, and ethylene glycol as monomers are used. Can be mentioned. Particularly preferred examples include polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylamide, poly(vinylpyrrolidone-vinyl acetate), and the like.

The mixing ratio of hydrophilic fine particles and hydrophobic group-containing fine particles in the reaction layer of the analytical element of the present invention is 5:1 to 60:1. Preferably, the ratio is about 10.l to 40:1. Further, the mixing ratio of the mixture of these two types of fine particles and the water-soluble binder is usually 1:1 to 20:1. The ratio is preferably about 3:1 to 12.1.

In the present invention, fluid samples in any form include solutions and colloidal solutions, but preferably fluid samples of biological origin, such as blood, plasma, serum, brain, cerebrospinal fluid, saliva, amniotic fluid, milk, urine, sweat, etc. Examples include meat juice.

A specific component in a fluid sample that can be measured according to 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 specific component. . That is, examples thereof include polypeptides, proteins, complex proteins, polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like.

Specific examples of these include, for example, Japanese Patent Application Laid-Open No. 63-4556
No. 2, No. 63-131062 or No. 63-13106
The substances described in No. 3 can be mentioned.

Substances that specifically bind to specific components in the vine fluid sample used in the present invention include antibodies, antigens, lectins, protein A, inhibitors of specific enzymes, etc., depending on the target to be measured. It is particularly preferred that the binding reaction of the binding substance is an antigen-antibody reaction. The origin of the antibodies used in the present invention is not particularly limited. Antigens can be administered to mammals, etc., or they can be obtained by conventionally known methods such as sodium sulfate precipitation, ammonium sulfate precipitation, or gel filtration using Sephadex gel. It can be purified and used by a suitable method, ion-exchange cellulose chromatography method, electrophoresis method, etc. (see Shunsuke Migita, ed., "Immuno Chemistry," Shinbun Shoten, pp. 74-88).

Alternatively, monoclonal antibodies may be produced by obtaining hybrid cells (hybridoma) from lung cells of a mammalian mammal (eg, a mouse) sensitized with an antigen and myeloma cells (myeloma).

In addition, these antibodies are IgG, IgM, IgA, Ig
D, IgE fractions can be used, or these antibodies can be treated with enzymes to produce Fab, Fab' or F(ab'
It may also be used in the form of active antibody fragments such as 1. Furthermore, these antibodies may be used alone or in combination. When an antibody or an antigen is used as a substance that specifically binds to a specific component in a fluid sample, the measurement principle of the analytical element of the present invention belongs to the immunoassay method, and its reaction types include the competitive method, two-antibody method, and Sand-Germany method. can be mentioned.

The hydrophilic fine particles constituting the reaction layer of the present invention are immobilized with a substance A that specifically binds to a specific component in the fluid sample, or a substance B that specifically binds to the substance A. There are two aspects. The substance B is an antibody against substance A, or substance A is an immunoglobulin G.
Examples include protein A when it is.

In a more preferred embodiment, the above-mentioned substance A, which specifically binds to a specific component in the fluid sample, is further bound to substance C, and the substance B, which specifically binds to substance C, is attached to hydrophilic fine particles. One method is to fix it. These substances B
Combinations of ゛ and C include: Enzyme Antibody Lectin: Nucleic acid Hormone: Substrate (product) Inhibitor Prosthetic group Coenzyme Allosteric effector Antigen Protein A Polysaccharide Glycoprotein Complementary base sequence Histone Nucleic acid polymerase Receptor Biotin : Avidin (biocytin) (streptavidin) (desthiobiotin) (oxybiotin), preferred are antibodies and antigens, or biotins and avidins, and more preferred are biotins and avidins.

These substances include those that do not bind or interact with the specific component in the fluid sample to be measured, the substance that specifically binds to the specific component, or the labeling substance used for measurement.

Methods for fixing and bonding the above-mentioned substances to hydrophilic fine particles are disclosed in JP-A-61-292060 and JP-A-62-90538.
, 62-90539, 63-45562, 63-
It is disclosed in No. 131062 and the like. In addition, a preferred method for producing the reaction layer of the present invention is to produce a coating solution using the hydrophilic fine particles of the present invention and a water-soluble binder in accordance with the method for producing a porous reaction layer described in the above-mentioned disclosure document, and Examples include a method of mixing and dispersing fine particles having a hydrophobic group in the above-mentioned ratio. Regarding the dispersion method and various preferable aspects (layer structure, shape, etc.) of the analysis using the reaction layer of the present invention, the disclosed examples of the above-mentioned disclosure documents can be applied as they are.

[Example] Next, the present invention will be specifically explained with reference to Examples.

1 Arashi Mei] (11p-aminophenylmercuric acetate (
Enzyme inhibitor) Preparation of immobilized Avicel Avicel (manufactured by Mukasei Co., Ltd., microcrystalline cellulose Avicel R3
F) 80g of 2.5M phosphate buffer fpH 12,018
Cyanogen bromide ao, O
After reaction for 20 minutes, the mixture was collected by filtration and thoroughly washed with water. 80 g of this Avicel was suspended in 950 ml of a 25% dimethyl sulfoxide aqueous solution containing 4.8 g of p-aminophenyl mercuric acetate, and stirred at room temperature for 20 hours. After filtering this and washing with dimethyl sulfoxide and pure water, 1M Tris-HCl buffer fpH8, 511,000ml
After stirring at room temperature for 20 hours, 0.2 MEDTA,
1NHCl, water, 40% DMF, 0.1% BSA, 8
After washing with M urea, 50 mM 2-mercaptoethanol, 8 M urea, 40% D liver, and water in this order, the product was dried to obtain p-aminophenylmercnock acetate-fixed Avicel.

(2) Preparation of avidin-immobilized Avicel Bovine serum albumin (Fraction V, manufactured by Arma, USA) 1. Og 015M Sodium Chloride Contains 0.01M
Dissolved in 330 ml of phosphate buffer (pH 7.4), added 3.0 ml of dimethyl formaldehyde solution containing 10.4 mg of biotin-N-hydroxysuccinimide ester (manufactured by Boehringa), and incubated at room temperature for 2 hours.
After 5 hours of reaction, the mixture was sufficiently dialyzed against the buffer solution and freeze-dried to obtain biotinylated bovine serum albumin.

Next, 90 g of Avicel (manufactured by Asahi Kasei Corporation) was added and suspended in 1800 ml of 2.5 M phosphate buffer (pH 12.0), and 45.0 g of cyanogen bromide dissolved in 550 ml of pure water was added to this under water cooling. In addition, after 20 minutes of reaction, it was collected and thoroughly washed with water. 90 g of this Avicel was added to an ice-water solution of 0.1 M carbonate bicarbonate (containing 0.15 M sodium chloride, 1
The mixture was suspended in 900ml and stirred at 4°C for 20 hours. Filter this and make pure water, 0.1M containing 015M sodium chloride.
Sodium bicarbonate aqueous solution, O, 0.1M sodium acetate buffer containing 15M sodium chloride (pH 4, 1
After washing alternately with 1M Tris-HCl buffer (p
H8,5] was suspended in 1200 ml and stirred at room temperature for 20 hours to block unreacted groups. After filtering this and thoroughly washing with water, use Avidin (manufactured by Oriental Yeast Co., Ltd.) 270
Contains 0.0 mg dissolved in 0.15M sodium chloride
The suspension was suspended in 450 ml of 5M phosphate buffer (pH 7.4), reacted at 4°C for 200 hours, collected, and lyophilized using a water washer to obtain avidin-immobilized Avicel.

(3) Preparation of β-galactosidase-labeled goat anti-human IgG Goat anti-human IgG (r chain specificity) (manufactured by Kappel, USA)
20 mg of O, 1M phosphate buffer (pH 6,512
, and 2.0 ml of N-(ε-maleimidocaproyloxy)succinimide (manufactured by Dojindo Laboratories) was added to this.
After adding 77 μl of 5 mg/ml dimethylformamide solution and reacting at 30°C for 20 minutes, it was purified using a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (fpH 6.0) containing 5 mM EDTA, and maleimidated. Goat anti-human IgG was obtained. Next, 10.5 mg/ml of β-galactosidase (manufactured by Toyobo Co., Ltd.) 0.1M#
! ! Add 32 ml of the solution containing 613.6 mg of maleimidated human Ig to 1.8 ml of acid buffer,
After reacting at ℃ for 45 hours, 175 μm of 0.1M 2-mercaptoethylamine was added and reacted at 30℃ for 20 minutes.
0.1M phosphate buffer containing 15&4 sodium chloride (p
The β-
Galactosidase-labeled goat anti-human IgG was obtained.

(4) Preparation of biotinylated sheep anti-human IgG antibody 5.8 mg of sheep anti-human IgG antibody (manufactured by Kappel, USA) was mixed with 0.1 M acid buffer containing 0.15 M sodium chloride (
Dissolve in pH 7,412,0 ml and add biotin-N to this.
- Dimethylformamide 5 containing 0.32 mg of hydroxysuccinimide ester (manufactured by Behringa)
Add 0.00μl and react at room temperature for 3.0 hours, then add 0.15M
The biotinylated sheep anti-human IgG antibody was obtained by thorough dialysis against a 0.01M phosphate buffer containing sodium chloride.

(5) Preparation of immunological analysis element Coating liquid (1) having the following composition was applied onto a 180 μm thick transparent undercoated polyethylene terephthalate film and dried to prepare a reaction layer.

Coating liquid + 1) p-aminophenyl 7-curitrate immobilized Avicel 24g Avidin immobilized Avicel
2.2g polyoxyethylene (10) octylphenyl ether 0.5g polyvinylpyrrolidone 0.83g5-10m-
4-chloro-3-indolyl-β-D-galactopyranoside 250mg3.3°i4.4°-biphenylene)-bis-(2,5-diphenyl 2H-tetrazolium chloride l
48mg n-butanol
15gANTARA 430 Kofulunyeon
0.5 g (Note) ANTARA 430 f vinylpyrrolidone/styrene copolymer, 40% emulsion, particle size 0.5 μm or less, manufactured by GAF Co., Ltd.) Furthermore, a coating liquid (2) having the following composition was applied to the reaction layer. A spreading layer was formed by coating and drying.

Coating liquid (2) Powder filter paper D (manufactured by Toyo Roshi Co., Ltd. 1 30.0g Triton
Cut into pieces of x 1.5 cm2 and use analytical element A (
Analytical element of the present invention).

Element B is an analytical element in which the amount of ANTARA 430 in the coating liquid (1) is changed to 0.2 g, and the coating liquid (2) is layered with the amount of ANTARA 430 in the coating liquid (1). Coating liquid (as Ig)
An analytical element in which 2) was layered was designated as element C. Furthermore, an attempt was made to layer the coating liquid (2) using 3 g of ANTARA 430 in the coating liquid fil, but it was not possible to form a film.

In addition, as a comparative analytical element for analytical elements containing these ANTARA 430, coating liquid (1) ANTARA 4
An analytical element was prepared by layering the coating liquid (2) with 0.05 g of the coating liquid (1).
Element E was an analytical element in which TARA 430 was removed.

(6) Measurement of human IgG 0,3M bis(2-hydroxyethyl)iminotris (hydroxymethyl) containing 1mM magnesium chloride, 5wt% collagen peptide A (derived from bovine bone, manufactured by Funakoshi Pharmaceutical), and 1wt% bovine serum albumin Methane buffer (pH 7,21) with 8 μg/ml of β-galactosidase-labeled goat anti-human IgG prepared in (3) and (4)
Biotinylated sheep anti-human IgG prepared with 32μg/
After mixing 70 μm of this solution with 10 μm of a human IgG solution dissolved in human serum minus IgG (manufactured by Kappel), 10 μm of this mixed solution was dropped onto the analytical elements A to E prepared in (5). After incubation at 37°C for 10 minutes in a sealed state, the reflection density at 546 nm was measured from the base side. The results are shown in the figure.

As is clear from the figure, the analytical elements A, B, and C of the present invention, in which a certain amount of fine particles having a hydrophobic group are added, are based on the difference in IgG concentration (reflection) compared to the analytical element of the comparative example. It can be seen that there is a large difference in concentration and that the test substance can be measured with higher sensitivity.It is also seen that there is a difference in effectiveness depending on the amount of microparticles with hydrophobic groups added1.
Almost no effect was observed with the amount added in analytical element B.

[Brief explanation of drawings]

The figure shows the IgG concentration and reflection density when IgG with different concentrations were measured using the analytical elements A, B, C, and D of the present invention to which fine particles having hydrophobic groups were added in different proportions, and the analytical element of the comparative example. FIG.

Claims (1)

[Claims] In an analytical element that measures a specific component in a fluid sample based on a binding reaction with a substance that specifically binds to the specific component,
The hydrophilic fine particles have a reaction layer containing a substance that specifically binds to the specific component or a substance that specifically binds to the substance, fine particles having a hydrophobic group, and a hydrophilic binder; and the fine particles having a hydrophobic group in a ratio of 5:1 to 60:1 on a weight basis.