JPH02143156A - Transfer sheet for pattern of electrophoresis fractionation - Google Patents

Abstract

PURPOSE:To obtain the sheet which transfers distinct and stable component fractionation spectra from electrophoresis fractionation patterns by providing one layer of a hydrophilic colloidal layer contg. a dye precursor and dye forming effect body on a liquid impermeable base. CONSTITUTION:The development medium to be deposited with the specific component fractionation is itself hydrophilic or is uniformly coated with a hydrophilic binder. The transfer sheet is also formed by coating the hydrophilic binder with the dye precursor (chromogen) and the dye forming effect body. The effect body presenting H2O2 is used for the dye forming effect and the chromogen forms the dye cooperatively with the effect body independently or together with the other compd. in the presence of the H2O2. The transfer sheet base is colorless or white and the binder for the development medium and the transfer sheet is so selected as not to hinder the peeling after the surface contact. The electrophoresis fractionation pattern of the specific component is formed on the development medium by this constitution to trigger the reaction combination on the medium and/or the transfer sheet by which the regular dye images are formed on the transfer sheet making the surface contact. The distinct and stable images are thus obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the analysis of biologically derived specimens using electrophoretic fractionation patterns, and specifically relates to the transcription and recording of electrophoretic fractionation patterns.

[Background of the invention]

Electrophoresis began to be used for the analysis of specimens of biological origin when interest was recognized in the electrophoretic fraction patterns of the Ti5elius apparatus and clinical phenomena. It has a different analysis mode from analytical elements that have been developed separately as a means of specifically and selectively detecting, quantifying, and analyzing specific components in a specimen, and develops the components of interest in a specimen as a fraction. However, electrophoresis, which allows for comparison and examination of changes in medical conditions, is becoming increasingly important as clinical research results accumulate.

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 used exclusively as a developing medium for developing the analyte sample into fractions by electrophoresis, but it is important to note that there is no diffusion of the liquid in the direction of the electric field of the formed electrophoretic carrier, or no convection of the liquid. , starch, agar, cellulose acetate, polyacrylamide, and other gel films have come to be 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 methods, color development methods include a method in which an enzymatic reaction caused on a developing medium such as an agarose film is colored with 4-aminoantipyrine, and a diformazane staining method with less fading property, etc. , is attracting a lot of attention because it allows a reliable diagnosis.

However, it is still not at a satisfactory level, and requires higher color density, a tone range 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 dye precursors. It is a leuco type, which dissolves at the same time as coloring, forming an inert pigment, and it is desired that the fractionation pattern does not collapse even when the developing medium is subjected to post-treatment, and the developing medium also has storage strength (damage resistance). Many problems remain to be solved, such as availability, convenience in storage, etc.

[Purpose of the invention]

The present invention avoids the inconveniences associated with the developing medium, and instead chooses a method in which the fractionated components are transferred from the developing medium to avoid the problems caused by the developing medium. To provide an electrophoretic fraction pattern transfer sheet capable of recording clear and stable component fraction spectra transferred from an electrophoretic fraction pattern without any restrictions and with good preservation in terms of quality and intensity.

In the present invention, to avoid confusion, the shape of the fraction formed on the development medium is called a fraction pattern, and the shape transferred and formed on the transfer sheet is called a fraction spectrum.

[Structure and effects of the invention]

The object of the present invention described above is to provide a system for transferring and recording electrophoretic fractionation patterns, in which at least one hydrophilic colloid layer containing at least a dye precursor and a dye-forming agent is provided on a liquid-impermeable support. This is achieved by using a transfer sheet.

In the present invention, the developing medium on which the specific component fraction is supported is a support that itself has hydrophilic properties, or a support that has a hydrophilic binder uniformly coated on the support.

The transfer sheet that is brought into contact with the developing medium is similarly coated with a hydrophilic binder containing a dye precursor (chromogen) and a dye-forming agent.

The chromogen according to the present invention is preferably non-diffusible and has a chin,
The chromogen may have a ballast group to provide diffusion resistance, such as couplers used in color photography, or it may be immobilized by providing a mordant layer.

Due to the formation of the dye, the bonding property to the ballast group or mordant layer is generally not lost, and the dye image formed in the fractionation spectrum that regularly corresponds to the fractionation pattern of the developing medium is disrupted due to the formation of the dye. There isn't.

In addition, the dye-forming agent that causes a reaction and links the specific component in the developing medium and the chromogen in the transfer sheet is a low molecular weight substance that can move freely in the hydrophilic binder, and is an enzyme. , redox substances, hydrogen peroxide produced in enzymatic reactions, and oxidized products of color developers produced in the color development of photographic couplers.
or a complex group thereof. That is, the chromogen is guided and linked to a color-forming reaction by oxidation or coupling through a primary reaction that occurs based on a specific component, or through a secondary reaction, a tertiary reaction, or the like.

Taking measurement noise into consideration, the support of the transfer sheet 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.

What is more important is that the developing medium binder and transfer sheet binder should be a combination of binders that are mutually compatible or have poor adhesion (for example, 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.

The present invention uses a developing medium and a transfer sheet, generates an electrophoretic fractionation pattern of a specific component in the developing medium, activates the reaction linkage in the developing medium and/or the transfer sheet, brings them into contact with each other, and applies the electrophoretic fraction pattern to the transfer sheet. A regular dye image is formed.

Both media can be configured to be optimal for the specific component 51, or can be configured to allow simultaneous detection and reaction of all 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 Riboprotein Lipase Triglyceride + 3 H, O Glycerol + 3 Fatty Acid 2
HtOz Decachrogen OD Pigment +4 H,0 ・Phospholibid Phosphorihi "Radonesfoolivase D Choline 0D 2HzCh Decachrogen Dye +4H
20 As the chromogen according to the present invention, hydrogen peroxide/POD
For coloring systems, hydrophobic ones are best.

For example, the combination of photographic color coupler and color developer has good stability of sensitivity dyes.

As the coupler, diffusion-resistant phenol compounds described in Japanese Patent Publication No. 63-37903 and diffusion-resistant pyrazolone compounds described in Japanese Patent Publication No. 63-37904 are particularly preferred.

In addition, JP-A-60-256056 (triallylmethane leuco dye), JP-A-57-16697 (triaryl imidazolyl leuco dye), JP-A-59-193353
(Diarylimidazolyl leuco dyes) can also be used. (However, diarylimidazolyl leuco dyes are subject to color stability.) These can be easily dispersed by a known dispersion method such as a direct dispersion method or an oil protect fraction method.

Furthermore, chromogens generally used in enzyme immunostaining methods and tissue staining methods can be used. Examples include 1) o-dianisidine or a salt thereof 2) o-tolidine or a salt thereof 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-naphthol, 4 -alkoxy-1
Examples include 7) tol derivatives such as naphthol, 7) leuco dyes such as leuco dyes such as leuco dyes such as leuco dyes such as 7) leuco dyes such as leuco dyes such as clean and leuco dyes.

Further, in the present invention, tetrazolium salts (Tetrazolium 5 alts) are preferably applied as the reduction coloring reagent, and NT 3-(p-1odophenyl)-2-(p-nit
rophenyl)-5-pheny-2Htetra
zolium chlorideTT 3-(4,5-Dimethyl-2-thiazol
yl)-2,5-diphenyi28 tetraz
oliua+ bromideNeo-Dingetrazo
lium Blue, Neo-TB (neotetrazolium blue) 3,3” (1,1'-Bipbenyi4,4'-
diy 1) bis(2,5-d 1phenyl-2
1tetrazolium chloride)Nit
rotetrazolium BIue, Nitro-
Trotetrazolium blue in TB) 3.3'-C3,3'-Dimethoxy-(1+
1'-biphenyl)-4,4'diyl)-bi
s(2-(p-nitrophenyl)-5-phe
nyl-2HtetrazoliuIIlchlori
de)TCtranitroLetrazoliuta
Blue, TNTB (tetranitrotetrazolium blue) 3.3'-1:3.3'-Dimatboxy-
(l l 1'-biphenyl)-4,4'-di
yl-bis(2,5-bis(p-nitrophe)
nyl)-2Htetrazolium chlori
de) TetrazoHutrr Blue, Ding B (tetrazolium blue) 3.3'-(3,3'-Dimethoxy-(1,l
'biphenyl)-4,4'-diyl)-bis
(2,5-diphenyl-2Htetrazoli
um chloride), etc.

There is no particular restriction on the amount of the chromogen added (1,01 to 1
．． 00a+ +wol/m', preferably 0.1-50
m s+ol/m".

Furthermore, the following are exemplified as operators that activate or cooperate with the reaction linkage to advance the coloring reaction.

Dehydrogenase system Cholesterol dehydrogenase Glycerol-3-phosphate dehydrogenase Glycerol dehydrogenase Choline oxidase (as dehydrogenase) Coenzyme NA, cotinamide dinucleoside (D) NADP (cotinamide dinucleoside phosphate) FAD (flavin adenine dinucleoside) ) Electron transfer agent Meldola's Blue (Mel Trouble-) 9
-Dimetbylavainobenzo-a -p
henazoxo (formerly umchloride) -Methoxy PMS (1-methoxy PMS
)1-Methoxy-5-methylphenaz
iniua+ methylsulfa nitrile PMS diaphorase (enzyme) These operators are appropriately used depending on the specific component to be analyzed.

The support for the transfer sheet of the present invention may be any conventionally known support as long as it is liquid-impermeable, and may be either light-transmissive or non-transparent. polymeric materials such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene;
Paper treated to make it impervious to liquid, natural polymeric materials such as fibers, inorganic materials such as glass, etc. can be used. Although its thickness is arbitrary, it is preferably about 50 to 1000 μm. It is also possible to improve the adhesion between the reagent layer and the support by using an undercoat layer in some cases on the support surface on which the reagent layer is provided.

The layer containing the dye precursor and dye-forming agent of the sheet according to the present invention is composed of a hydrophilic binder, and the binder includes gelatin, gelatin derivatives such as phthalated gelatin, hydroxyethyl cellulose, carboxymethyl cellulose sodium salt, etc. water-soluble cellulose derivatives, polyvinylalcohol, polyacrylamide, polymethacrylamide, poly(mono- or dialkyl-substituted)
Examples include acrylamide, poly(mono- or dialkyl-substituted) methacrylamide, and water-soluble copolymers thereof.

Furthermore, it is also possible to provide at least one layer containing an enzyme that converts the target specific component into a diffusible substance on the uppermost layer of the transfer sheet. The binder in this layer immobilizes the sheet and the target specific component; in order to bring the enzyme into contact with the specific component efficiently when the support is brought into contact, a binder that provides a certain degree of diffusivity is preferably used in addition to the hydrophilic binder described above. Examples include layers of fibrous, porous, particle-bound structures, and the like.

The various layers of these sheets are sequentially laminated on the support according to the present invention by appropriately selecting a slide hopper coating method, an extrusion coating method, a dip coating method, etc. conventionally known in the photographic industry according to the desired configuration. This allows a layer of arbitrary thickness to be applied.

〔Example〕

The present invention will be specifically explained with reference to Examples.

Example-I Deionized gelatin 10g/m'P
OD 5000U/ln”
Diethyl phthalate 5.3g/Il+”
1-(2,4,6-trichlorophenyl)-3-(2-
Chloroffdabcylsuccinimideanilino)-5-pyrazolone 6.7g/a21.8M potassium phosphate buffer (pl(7,2)38.0g/a' Alkanol XC (Dupont) 0.15g/+"
Bis(vinylsulfonylmethyl)ether 0.36g/
It was coated onto a white polyethylene terephthalate support coated with an undercoat to a thickness of 180 .mu.m to give a sheet (1).

High lipid serum (total cholesterol 360 mg/dQ, triglyceride 4
80+ag/dL Phospholipid 28Qtmg/d
It was Q. ), create a dilution series of ×1.0, ×17
2, ×l/4, and XI/8 were plotted on the membrane.

A reaction solution was prepared by adding the following sets of reagents to a 0.3M potassium phosphate buffer containing 50mM of N,N-diethyl-3-methanesulfonamidoethyl-4-aminoaniline/p-toluenesulfonic acid. .

Cholesterol oxidase for total cholesterol IU/++Q cholesterol esterase IU/-1I2 Lipoprotein lipase for triglyceride O,? U/m(I
L-σ-glycerophosphate oxidase 0.5U
/raQ For Phos7 Oribit* Supho IJ Base D 1.2U/m
i2 choline oxidase 0-3U 1m
Q After impregnating the transfer sheet of the present invention with the above reaction solution, it was superimposed on a cellulose acetate film, incubated at 37°C for 115 minutes, and then the cellulose acetate film and sheet were peeled off, washed with water, dried, and then transferred to a Konica photodensitometer P. D
The reflection density was set to 5 using A-65 (manufactured by Konica Corporation).
Measurement was carried out using a 46H filter.

As described above, good linearity was demonstrated.

Example 2 A layer having the following composition was coated on a transparent polyethylene terephthalate support having a thickness of approximately 180 μm.

Gelatin 10g/m"Diaphorase 60001J/m"3.3
'-(1,1'-bisphenyl-4,47-diyl)-
Bis(2,5-diphenyl-2H tetrazolium chloride) 23Qm moQ/m”T
rito mouth ■X-100 (ROHM Entnos Co., Ltd.)
0.3g/+o” Bis(vinylsulfonylmethyl)ether 0.36g/
m2 This sheet was designated as the sheet (2) of the present invention.

Furthermore, cholesterol esterate 0.3U/ml in 0.3M Tris-HCl buffer (pH-8,6) 12 cholesterol dehydrogenade 0-20U/a+Q N A D
Prepare a solution dissolved to give moQ/mQ, and then add the high-fat serum prepared in Example-1 to the cellulose acetate-1 membrane for electrophoresis prepared in Example-1. After plotting, the sheet was impregnated with the enzyme solution and layered with the sheet (2) of the present invention, incubated at 37° C. for 15 minutes, and evaluated in the same manner as in Example-I, showing good linearity.

〔Effect of the invention〕

The method of the present invention does not require adjustment of the staining solution, and the generated dye is transferred to the stabilizer sheet, compared to the conventional method of dipping a support on which a specific component is immobilized into a dyeing solution to create a colored pattern on the support. It has excellent mechanical strength, allows for long-term storage of patterns, allows the analysis of various substances using the same sample, allows the use of color strips that cannot be dissolved in staining solutions, is advantageous in terms of sensitivity, and has a wide range of color tones. There are many advantages such as a wider range of choices.

Claims (5)

[Claims] (1) In a system for transferring and recording electrophoretic fractionation patterns, a transfer sheet comprising at least one hydrophilic colloid layer containing at least a dye precursor and a dye-forming agent on a liquid-impermeable support. (2) The transfer sheet according to claim 1, wherein the dye-forming agent is an agent exhibiting a peroxidizing effect. (3) The dye precursor is a compound that produces a dye alone or together with other compounds in the presence of hydrogen peroxide in cooperation with the agent exhibiting a peroxidizing effect. 2. The transfer sheet described in 2. (4) The transfer sheet according to any one of claims 1 to 3, wherein the dye-forming agent contains a dehydrogenase. (5) The transfer sheet according to any one of claims 1 to 4, wherein the dye precursor is a tetrazolium salt.