JPH02143155A - Analysis of specific component - Google Patents

Abstract

PURPOSE:To facilitate diagnosing of recording by bringing a transfer medium into which the chromogen of a nondiffusion state is incorporated into surface contact with a matrix medium on which a specific component derived from an organism is fixed and deposited and making analysis by using the reaction combination elements moving between the specific component and the chromogen facing each other via the contact surface at the time of analyzing the specific component. CONSTITUTION:Carriers, such as cellulose acetate and nitrocellulose, which are generally used in an electrophoresis method are used for the matrix medium to be fixed and deposited with the specific component. A base coated with a hydrophilic binder into which the chromogen is incorporated is used as the transfer medium to be brought into surface contact therewith. The spots or sepn. patterns of the specific component are generated in the matrix medium by using the matrix medium and the transfer medium formed in such a manner. The generated reaction combination elements are formed as a regular dye images on the transfer medium. The need for preparing a color forming liquid is eliminated and the formed dyes are stabilized; in addition, the long-term preservation of the pattern is possible according to this method.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for analyzing specific components derived from living organisms.

[Background of the invention]

Although sophisticated instrumental analyzers are used to analyze specific components derived from living organisms, various analytical element methods are used because they are simple, rapid, and inexpensive.

As one typical example, an analyte sample is spotted on a medium layer that also contains a reagent and a label that specifically reacts with a specific component,
An analytical element method that specifically detects and quantifies a specific component by its color development, fluorescence, etc., or a method in which the sample is dotted on one side of the medium, and as the developing solvent rises due to capillary action, the sample components are pulled up and developed into fractions. Examples include chromatography in which detection is performed by staining, etc., and electrophoresis in which an electric field is applied between both sides of the medium in the chromatography. Electrophoresis has higher practical value than chromatography because it has more freedom in separating fractions.

These methods often use color development or coloring as a means of revealing spots or fractions of specific components.

Many techniques have already been disclosed regarding the analysis element on which the sample body fluid is applied.

However, the detection medium related to electrophoresis has not yet been sufficiently studied.

The electrophoresis method began to be used for the analysis of specimens derived from living organisms when interest was recognized in the electrophoretic fraction patterns of the Ti5elius device and clinical phenomena. It has a different analysis mode from the analytical elements that are being developed, and it develops the components of interest in the specimen as fractions, which can be compared and examined with changes in disease conditions, etc., and is useful for clinical research. With the accumulation of results, the importance of this field is increasing.

The method currently used for clinical analysis is zone electrophoresis, which is an ideal method for clinical analysis because it uses simple equipment, is inexpensive, and can be applied to minute samples.

In the early days, filter paper was exclusively used as a developing medium for developing the analyte sample into fractions by electrophoresis, but starch, agar,
Gel films such as cellulose acetate and polyacrylamide are now being used.

To search and quantify the developed fractional components, an extraction method, a determination method based on shape, an area method, a color method, etc. are used in addition to the developed position of the fraction.

Among these, color development methods include a method in which an enzymatic reaction that occurs on a developing medium, such as an agarose film, immersed in a staining solution is colored and stained with 4-aminoantipyrine, and a diformazane staining method that is less likely to fade has been developed. , the ability to quickly and reliably make a diagnosis based on clear fractionation patterns has attracted much attention.

However, it is still not at a satisfactory level, and requires higher color density, a tone gamut that does not include noise in density determination, the ability to print fractional patterns on color photographs, etc., no contamination of the developing medium, and the ability to use color originals. It is a leuco type, which forms a poorly soluble and inert pigment at the same time as coloring, and it is required that the fractionation pattern does not collapse even when the developing medium is subjected to post-treatment. ),
Many problems remain to be solved, such as ease of storage. These problems are common to analytical elements, chromatography, and the like.

[Purpose of the invention]

The present invention avoids the troubles associated with spotting and developing media, and instead isolates and transfers images that regularly correspond to specific components in spotting spots and developing fractions to other media. , by forming a dye image corresponding to the regularity (regular dye image), we have selected a means to overcome the obstacles caused by the medium, and in accordance with this means, the object of the present invention is to: (1) Restrict the coloring material to be used; (2) without being
The purpose of the present invention is to provide clear, stable regular dye images with good storage stability, (3) convenience for repeated observation, and (4) records with good damage resistance for diagnostic purposes.

In the present invention, in the case of electrophoresis or chromatography, in order to avoid confusion, the shape of a fraction of a specific substance is called a fractionation pattern, and the transferred shape is called a fractionation spectrum.

[Structure and effects of the invention]

The object of the present invention is to place a transfer medium containing at least a chromogen in a diffusion-resistant state in contact with a matrix medium fixedly supporting a specific component, and the specific component facing the specific component across the contact surface. This is achieved by a method for analyzing a specific component that forms a dye on a transfer medium by a reactive linker that acts between both chromogens, and a method for analyzing a specific component that peels off the transfer medium on which the dye has been formed and uses it for detection. .

In the present invention, the matrix medium on which the specific component is immobilized can be a carrier generally used in electrophoresis or a carrier used for antigen immobilization in immune reactions, such as cellulose acetate, nitrocellulose,
Examples include membranes such as polyvinylidene fluoride and nylon, gel carriers such as polyacrylamide, agar, and starch, gel carriers such as TLC plates, and glass, metal, fiber, and filter paper in the form of plates and sheets. Furthermore, biological tissues themselves such as tissue sections can also be used as the matrix medium.

The transfer medium brought into contact with the matrix medium preferably has a structure in which a binder containing a chromogen is coated on a support. As the binder, a hydrophilic binder is preferred.

The chromogen in the diffusion-resistant state according to the present invention may have a ballast group on the chromogen, such as a coupler used in color photography, to make it diffusion-resistant, or may be provided with a mordant layer. It may also be by means of immobilization.

Further, the diffusion resistance required in the present invention is sufficient as long as it does not interfere with actual operation, and can be determined experimentally from a practical standpoint.

Bonding to the ballast group or mordant layer is generally not lost due to the dye formation, and the dye formed does not diffuse and disrupt the regular dye image.

In addition, the reactive linkage that acts between the specific component in the matrix medium and the chromogen in the transfer medium is a low molecular weight substance that can move freely in the binder having a capillary structure, and is a substance with a low molecular weight that can move freely in the binder having a capillary structure. oxidized products of color developing agents, NADHz, FADHx generated during color development of photographic couplers.
Examples include reduced coenzymes such as

That is, as a primary reaction product (e.g. hydrogen peroxide, NADHZ, FADL) generated based on a specific component or a secondary reaction product (e.g. oxidized color developer) from the reaction product, the chromogen The chromogen is led to a color-forming reaction by oxidation or coupling, or by reducing the chromogen.

Taking measurement noise into consideration, the support of the transfer medium is prepared in a transparent color, such as colorless, for transmitted light measurement, and in a preferable base color, such as white, for reflected light measurement.

More importantly, the matrix media binder and the transfer media binder should be a combination of binders that are not mutually compatible or have poor adhesion (e.g., gelatin and agar) so that complete and smooth peeling can be achieved without causing peeling problems after contact. etc.), or a liquid-permeable release layer is provided on at least one side. Alternatively, a liquid layer may be added to facilitate peeling when the surfaces are brought into contact.

The present invention uses a matrix medium and a transfer medium prepared as described above, generates a spot or fractional pattern of a specific component on the matrix medium, and includes a means for generating the reactive linkage on the matrix medium and/or the transfer medium. The two are brought into contact and a regular dye image is formed on the transfer medium.

Both media can be configured to be optimal for specific acid fractionation, or can be configured to enable simultaneous detection and reaction of multiple components.

Next, an example in which the present invention is applied to an oxidase system will be shown below.

Cholesterol Cholesterol Esterase Cholesterol Ester Cholesterol + Fatty Acid Triglyceride JJ Bobrotiin Lipase Triglyceride + 3H 30 Glycerol + 3 Fatty Acids 2H
10! Decachrogen OD Pigment +4H30 ・Phospholibid Phos 7 Years Pid Phospholipase D Choline 2Hzb OD Pigment +4H80 OD 2 Hz O to Decachrogen Pigment +
4HjO Also, the following is an example of applying the present invention to a dehydrogenase system: cholesterol triglyceride g bobrotiine lipase triglyceride + 3H80 glycerol + 3 fatty acid dihydroxyacetone phosphate 1NADH! NADH, Decachromogen diaphorase dye + NAD Phospholibid Phosphoribit Phospholipase D Choline The chromogen according to the present invention is preferably a hydrophobic one. As a chromogen in a hydrogen peroxide/POD coloring system, for example, a photographic color coupler, a color developer combination has a sensitivity,
The stability of the dye is also good.

Other examples include triarylmethane leuco dyes described in JP-A No. 60-256056, triaryl imidazolyl leuco dyes described in JP-A-57-16697, and JP-A-59-1.9.
Diarylimidazolyl leuco dyes described in No. 3353 can also be used.

Furthermore, chromogens generally used in enzyme immunostaining methods and tissue staining methods can be used. Examples include 1) o-dianisidine or its salt 2) o-) lysine or its salt 3) 3-amino-9-ethylcarbazole 4) Benzidine, 3.3'-diaminobenzidine, 3.3', 5 ．．
Benzidine derivatives such as 5'-tetramethylbenzidine or salts thereof 5) Combination of 4-aminoantipyrine or derivatives thereof or salts thereof and phenol or naphthol or derivatives thereof 6) 4-chloro-1-su7thol , 4-alkoxy-1
- Naphthol derivatives such as naphthol 7) Combination of aromatic primary amine compound and phenol compound disclosed in JP-A-61-150723 8) Aromatic primary-class disclosed in JP-A-61-21699 Combination of amine compound and active methylene compound 9) Examples include leuco dyes such as leucomacide green and leucophenolphthalene.

As the chromogen in the reduction coloring system, compounds known as tetrazolium salts are generally used. An example is NT 3- (p-1odopheny 1)-2-(p-n
iLropheny I)-5-phany
12HLeLrazolium chlorideTT 3-(4,5-Dimethyl-2-Lhiazol
yl)-2,5-diphenyi28 Letraz
olium bromtdeNeo-Tetrazol
ium Blue Neo-TB (neotetrazolium blue) 3.3'-(1,l'-Biphenyl-4,4'-
diyl)bis(2,5-diphenyl-2Ht
etrazolium chloride)NiLro
teLrazolium Blue, N1tro-TB
Trotetrazolium blue) 3.3'-(3,3'-Dimethoxy-(1,1
'-biphenyl)-4,4'diyl)-bis
(2-(p-nitrophenyl)-5-phen
yl-2Htetrazolium chloride
) TetraniLrotetrazolium Blue
e, TNTB (tetranitrotetrazolium blue) 3
．． 3”(3,3'-Dimethoxy-(1,l'-
bjphenyl)-4,4'-diyl-bis(2
,5-bis(p-nitrophenyl)-2Ht
Tetrazolium Blue TB (tetrazolium blue) 3.3'-(3,3'-Dimethoxy-(1, l
'biphenyl)-4,4'-diyl)-bis
(2,5-diphenyl-2Htetrazoli
um chloride), etc.

The amount of the chromogen added is not particularly limited, but is 0.01 to 10.
0mmol/l! l”, preferably O, 1-50m
mol/m".

The method of adding the chromogen according to the present invention to the liquid for forming the transfer medium according to the present invention can be appropriately selected depending on the chemical structure of the chromogen. For example, various methods can be used, such as a method of dissolving and adding in water, an aqueous buffer solution, an organic solvent, etc., a solid dispersion method, a latex dispersion method, an oil-in-water emulsion dispersion method, and the like.

The oil-in-water emulsion dispersion method can be applied to a method of dispersing hydrophobic additives such as couplers, which are conventionally known in the photographic industry, and are usually dissolved in a high-boiling point solvent and/or a low-boiling point solvent, and an anionic surfactant. And/or it can be mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing a nonionic surfactant, and emulsified and dispersed using a high-speed rotation mixer, colloid mill, flow jet mixer, ultrasonic dispersion device, etc. and used.

Operators that generate reactive linkages or proceed with coloring reactions in cooperation with them include hydrolytic enzymes such as cholesterol esterase, riboprotein lipase, and phospholipase, cholesterol oxidase, choline oxidase, glycerol oxidase, l-σ-glycerophosphate oxidase, Oxidizing enzymes such as peroxidase and catalase, phosphorylating enzymes such as glycerol kinase, cholesterol dehydrogenase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase,
Dehydrogenases such as choline oxidase (acts as a dehydrogenase), formaldehyde dehydrogenase, diaphorase, Meldola's Blue (9-Dime)
thYlaminobenzo-g-phanazo
xonium chloride), 1-Methoxy
PIJS (1-Methoxy-5-methyl
phenaziniu+++5etbylsu! fat
e) Electron transfer agents such as PMS and the like.

These operators are used appropriately depending on the specific component to be analyzed.

These operators may be contained in the chromogen-containing layer of the transfer medium, or in other layers, or may be added from the outside when the matrix medium and the transfer medium are in contact with each other. , for example, may be supplied in a solution state.

It is preferable that an agent, such as peroxidase or diaphorase, which promotes a coloring reaction in cooperation with a reactive linker, be contained in the layer containing the chromogen.

A functional layer containing various functional elements may be further provided on the side facing the transfer medium. It is preferable that the active layer contains an operator that generates a reactive linkage, such as a hydrolytic enzyme such as cholesterol esterase, an oxidizing enzyme such as cholesterol oxidase, and a dehydrogenase such as cholesterol dehydrogenase.

When the binder of this working layer is brought into contact with the matrix medium, it is preferable to use a binder that provides a certain degree of diffusivity in order to efficiently bring the specific component into contact with the operator, especially the operator that generates the reaction linking element, and to form the binder layer structure. Other examples include layers with fibrous, porous, and particle bond structures.

In the present invention, known mordants can be used for the mordant layer provided as necessary.

Examples include quaternary ammonium salt-containing polymers described in Japanese Patent Publication No. 57-24537 and tertiary amine-containing polymers described in JP-A-54-124726.

The mordant layer may contain various agents and/or chromogens.

Binders for the transfer medium according to the present invention include gelatin, gelatin derivatives such as phthalated gelatin, water-soluble cellulose derivatives such as hydroxyethyl cellulose and carboxymethyl cellulose sodium salt, polyvinyl alcohol,
Examples include polyacrylamide, polymethacrylamide, poly(mono- or dialkyl-substituted) acrylamide, poly(mono- or dialkyl-substituted) methacrylamide, water-soluble copolymers thereof, agar, starch, and the like.

The support for the transfer medium of the present invention may be a conventionally known support, such as a polymeric polymer material such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene, a natural polymer material such as paper or fiber, or an inorganic material such as glass. Materials and the like are selected and used depending on the aspect of the invention.

Its thickness is arbitrary, but preferably about 50 to 1000μ
It is m.

It is also possible to improve the adhesion between the binder and the support by using an undercoat layer on the surface of the support on which the binder layer of the medium is provided, as the case may be.

The various layers of the transfer medium can be sequentially laminated on the support according to the present invention using a slide hopper coating method, an extrusion coating method, a dip coating method, etc., which are conventionally known in the photographic industry and are appropriately selected according to the desired configuration. A layer of any thickness can be applied.

Compared to the conventional method of immersing a matrix medium in which a specific component is immobilized in a staining solution to create a colored pattern on the matrix medium, the method of the present invention does not require the preparation of a staining solution, the produced pigment is stable, and the dye is stable. It has excellent mechanical strength, allows patterns to be stored for a long time, allows analysis of various substances using the same sample, allows the use of chromogens that cannot be dissolved in staining solutions, is advantageous in terms of sensitivity, and improves color tone. This has many advantages, such as a wider range of choices.

〔Example〕

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

Example 1 (1) Creation of transfer medium A layer containing a reagent having the composition shown in Table 1 and a chromogen shown in Table 2 was coated on a transparent undercoated polyethylene terephthalate support with a film thickness of 180 μm, and Transfer sheets ① to ② shown in -2 were prepared.

Table-1 HEPES book
5g/m”NaOH0.33
g/m” Peroxidase 1.0,0OOU/
m” gelatin 21g/m
2 Dimedone 0.3g/m
”Sodium triisopropylmethacrylsulfonate
0.7g/m”1.2-bis(vinylsulfonyl)ethane zero HEPES 0.2g/m2 N-2-Hydroxyethylpiperazin
e-N'4-ethanssulfonic aci
dTable-2 Sheet ■ Compound 1 ■ Compound 2 1,. 5g/+'1.5g/m"
■ 4-Ethoxy-1-naphthol 1.5 g/m” Compound 1 Compound 2 H (2) Cholesterol measurement Add cholesterol esterase to phosphate buffered saline (pH 7.4: hereinafter referred to as PBS) and cholesterol oxidase to 112 IU/l. Enzyme solution A was prepared by adding enzyme solution A to a concentration of IU/mQ.Furthermore, 0.2 mg 7 m (l Enzyme solution B was prepared.

Control serum (total cholesterol (ill, 140
+mg/dI2) was serially diluted with PBS, and 2 μα
plotted. After this nitrocellulose membrane was immersed in enzyme solution A or B for 1 second, the prepared transfer sheet was adhered thereto, and the membrane was kept at 37° C. and allowed to stand still. When enzyme A is used, transfer sheets ■ and ■ each have a concentration of 100 mg/dL 3
Cholesterol of 0+ag/dff was detectable. In addition, when enzyme solution B is used, sheet ■ is 0.3 Pa
Cholesterol was detectable at 3 mg/dQ in Sheet ■ and 1 mg/dα in Sheet ■.

Example 2 Control serum was diluted to 1/30 with PBS and plotted on a nitrocellulose membrane in 2 Pa increments.

After being immersed in enzyme solution B for about 1 second, a sheet prepared in the same manner as in Example 1 was brought into close contact with the sample, and after being kept at 37° C. for 30 minutes, it was allowed to stand at room temperature in a bright place for 4 weeks. The colored spots did not change even after 4 weeks.

Comparative Example (1) 3ymg of 4-chloro-1-naphthol was dissolved in methanol, and enzyme solution B was added with 5filQ and 5U/m (1 part of horseradish peroxidase 1II112,
A staining solution was prepared. 3. Place the nitrocellulose membrane in the staining solution.
It was immersed at 7°C for 30 minutes to develop color. The membrane was then washed with water and dried. The colored spots disappeared after being allowed to stand for 4 weeks at room temperature in a bright place. Note that when Compound 1 and Compound 2 of Example 1 were used as chromogens, a coloring solution could not be prepared due to precipitation. The same result was obtained even when an organic solvent compatible with water other than methanol was used.

Example 3 Acarose electrophoresis system (manufactured by Ciba Corning)
Using agarose film as a matrix medium, 3μ of normal human serum was electrophoresed.

After electrophoresis, enzyme solution B prepared in the same manner as in Example 1 was sprinkled, and a transfer sheet (3) prepared in the same manner as in Example 1 was adhered. When the sheet was allowed to stand at 37° C. for 30 minutes, the colored spectrum of cholesterol appeared on the sheet. The sheet was peeled off, washed with water, and then dried. The color spectrum on the sheet remained stable even during long-term storage.

Example 4 Using a Titan electrophoresis system (manufactured by Helena Laboratories), using a cellulose acetate membrane as a matrix medium,
Normal human serum was run.

Phospholipase D was dissolved in PBS at a concentration of 20/mff and choline oxidase was dissolved in PBS at a concentration of 2 U/mQ to prepare an enzyme solution. After immersing a cellulose acetate membrane in this for one second, a transfer was prepared in the same manner as in Example 1. The sheet ■ was placed in close contact. When the sheet was left standing at 37° C. for 30 minutes, a phospholipid color spectrum appeared on the sheet. The sheet was peeled off, washed with water, and then dried. The color spectrum on the sheet remained stable even during long-term storage.

Example 5 A reagent layer having the composition shown in Table 3 was applied onto the transfer sheet (2) prepared in Example 1 to prepare a transfer sheet (2).

Table-3 HEPE5
5g/m'NaOH0.33g/m" Cholesterol oxidase 6.000[1/
m"cholesterol esterase 6.0OOL
I/m' Sodium triisopropylnaphthalene sulfonate 0.3 g/m' Gelatin 2.0 g/m' An agarose film on which serum was electrophoresed in the same manner as in Example 3 was moistened with pure water as a matrix medium, and a sheet ■ were placed in close contact.

Install for 30 minutes at 37℃! Around this time, the colored spectrum of cholesterol appeared on the sheet.

The sheet was peeled off from the agarose film, washed with water, and then dried. The color spectrum on the sheet remained stable even during long-term storage.

Example 6 (2) Preparation of transfer medium A layer containing the reagent and chromogen having the composition shown in Table 4 was coated on a transparent subbing polyethylene terephthalate support having a film thickness of 180 μm to prepare a transfer sheet (2).

Further, a reagent layer having the composition shown in Table 5 was applied onto the sheet to prepare a sheet (2).

Table-4 Neotetrazolium Blue 0.8g/m'Diaphorase 2,0OOU/m"
Glycerol dehydrogenase 20, 0OOIJ
/m ”gelatin 25g/
m'1-octylphenoxypolyethoxyethanol 0
．． 1g/m' 1.2-bis(vinylsulfonyl)ethane 0.1g/m
” Table-5 Riboprotein lipase 8.00011/m
'NAD 1.3g
/m"p-octylphenoxypolyethoxyethanol 9 g/m" Gelatin 20g/+n22
) Measurement of triglyceride As in Example 3, an agarose film carrying the fractionation pattern of serum was used as the matrix medium. In the case of transfer sheet ①, the matrix is immersed in a pH 10 buffer and the sheet is brought into close contact with ①. Transfer sheet ■
In the case of , the matrix medium was immersed for 1 second in a pH 1o buffer containing lipoprotein lipase at a concentration of 1.5 U/mQ, N A D at a concentration of 3 mg/mQ, and then
The sheets were pressed together. When the sheets were left undisturbed at 37° C. for 30 minutes, a color spectrum of triglyceride appeared on the sheets ① and ②.

The sheet was peeled off from the agarose film, washed with water, and then dried. The color spectrum on the sheet remained stable even during long-term storage.

Claims (2)

[Claims] (1) In a matrix medium that fixedly supports specific components,
A transfer medium containing a chromogen in at least a diffusion-resistant state is brought into contact with the transfer medium, and a dye is formed on the transfer medium by a reactive linkage that acts between the specific component and the chromogen that face each other across the contact surface. Analysis method for specific components. (2) A transfer medium containing at least a chromogen in a diffusion-resistant state is brought into contact with a matrix medium that fixedly carries a specific component, and a transfer medium is formed between the specific component and the chromogen that face each other across the contact surface. A method of analyzing a specific component by peeling off a transfer medium that forms and carries a dye using a reactive linker that acts and is used for detection.