JPH03251765A - Biological analytical reagent composition and multilayer analytical element - Google Patents

Abstract

PURPOSE:To obtain an excellent biological reagent and a multilayer analytical element generating no lowering of sensitivity by using a composition containing a specific compound and/or other specific compound and further containing a specific anion species and a multilayer element containing said composition. CONSTITUTION:A compound represented by formula (I) and/or a compound represented by formula (II) and, further, an anion seed such as phosphate are contained in a multilayer element wherein a layer containing a hydrophilic polymer and a porous developing layer are successively laminated to a light permeable support. In the formulae, Y is a 2 - 4C straight chain alkylene group which may have a substituent, R<1> is a 1 - 4C alkyl group or a hydroxyethyl group, R<2> is a 1 - 4 alkyl group which may have one or two OH group or one phenyl group, W is H or a nitro group and Z is a 4,5-dimethyl-2-thiazolyl group when W is H and 4-iodophenyl group when W is a nitro group, R<3> is a methoxy group and may be H when W is H and X<-> is Cl<-> or Br<-> being a counterion setting off charge in a compound. The substitution position in a benzene ring represented by formula III containing R<1>, R<2> N and OY is a 3- or 4-position.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to the analysis of biological samples.

(Conventional technology) Traditionally, biological samples, eg tissues, blood cells.

Components contained in blood (plasma, serum), urine, etc. are analyzed chemically, biochemically, and immunologically.

In particular, when a color reaction is used to detect the results, there are fewer operational restrictions and it becomes easier.

Therefore, color reactions are often used for detection in tissue staining, especially immunohistochemistry plotting, and in clinical chemistry, especially dry chemistry using multilayer analytical elements.

As for the characteristics required for these, it is necessary that the produced dye has low solubility in a solvent.

In immunohistological staining, a method of staining using horseradish peroxidase (HRP) as a labeling enzyme and diaminobenzidine as a chromogen is often used.

(Problems to be Solved by the Invention) In the above-mentioned immunohistological staining, after an antigen-antibody reaction is performed on a tissue section, in the direct antibody method, a coloring reaction is performed using an enzyme labeled with the antibody used. .

In the indirect antibody method, an enzyme is reacted with a labeled secondary antibody, and then an enzymatic reaction is performed to form a dye.

The dye stains the area where the antigen is present and does not elute to other areas.
In addition, it is necessary that the organic solvent does not dissolve in the dehydration and clearing steps. Furthermore, DAB, which is often used for this purpose, is carcinogenic and carries great risks. Also in the blotting method, dissolution of the dye formed on the membrane by the solvent impairs analysis accuracy.

Also, in common with these, the storage stability of the dye itself is also important. The diaminobenzidine and tetrazolium salts used for the above purpose are carcinogenic, while the latter poses problems in terms of solubility and storage stability.

On the other hand, in a multilayer analytical element, when the generated dye elutes and moves to the porous development layer without staying in the detection layer, the transferred dye is hidden by the development layer, and the reflection density that should be obtained cannot be obtained, resulting in sensitivity. decreases. This problem also occurs when a poorly soluble formazan dye is used in a multilayer analytical element using a solution method.

(Objective of the Invention) The object of the present invention is to eliminate the above-mentioned drawbacks and provide an excellent biological reagent composition, and another object is to provide an excellent multilayer analytical element that does not cause a decrease in sensitivity. It is in.

(Means for Solving the Problems) As a result of extensive studies in line with the above objectives, the objective of the present invention is to provide a compound represented by the following general formula [I] and/or a compound represented by the general formula [11]. Furthermore, it is achieved by a biological analysis reagent composition comprising a specific anionic species and a multilayer analysis element containing the reagent composition.

General formula [I] General formula CI [) In the formula, Y is a linear alkylene group having 2 to 4 carbon atoms,
It may have a substituent, and one hydroxyl group may be present as the substituent.

R1 is an alkyl group having 1 to 4 carbon atoms, or a hydroxyethyl group %R'' is an alkyl group having 1 to 4 carbon atoms;
It may have two hydroxyl groups or one phenyl group.

W is a hydrogen atom or a nitro group.

Z is a 4,5-dimethyl-2-thiazolyl group when W is a hydrogen atom, and a 4-iodophenyl group when W is a nitro group.

Further, R3 is a methoxy group, but when W is a hydrogen atom, it may be a hydrogen atom.

Xe is an anion that cancels out the charge in the compound, and is a chlorine ion or a bromine ion.

1 Also, the substituent position 2 in the benzene ring of -0Y-N-R' The position is 3rd or 4th place.

The multilayer analytical element of the present invention is a multilayer analytical element constructed by sequentially laminating at least a layer containing a hydrophilic polymer and a porous spreading layer on a light-transmitting support.

The compound according to the present invention is disclosed in Japanese Patent Publication No. 59-39430.
No. 60-3396, it is a product in which a quaternary ammonium salt is introduced into a tetrazolium salt, and as described in the above specification, a tetrazolium salt and Introducing a quaternary ammonium salt, which is a water-soluble group, to the formazan dye,
The purpose is to impart water solubility and prevent contamination of the reaction vessel during solution reaction.

In accordance with the purpose of the present invention, the present invention has been developed in the course of investigation that when the above-mentioned compound reacts and changes into a formazan dye, the solubility disappears not only in water but also in organic solvents in the presence of specific anion species. This is based on recent findings, especially when ethanol (used in the dehydration step in immunohistological staining)
It has been found that it has the property of not being dissolved at all in xylene used in the clearing process.

Preferred anionic species specified in the present invention include, for example, a series of compounds known as phosphoric acid, carbonic acid, acetic acid, and gas buffers, such as N-(2-acetamido)=2-aminoethanesulfonic acid, N-(2-acetamido)iminodeacetic acid, N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, 3-cyclohexylaminopropanesulfonic acid , 3-N-cyclohexylthymine-2-hydroxypropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonic acid, 3-(N,N
-bis(2-hydroxyethyl)aminoco-2hydroxypropanesulfonic acid, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid, N-2-e droxyethylpiperazine-N/-2-ethanesulfone acid, N-2-hydroxyethylpiperazine-N'2-hydroxypropane-3-sulfone a, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, 3-(N-morpholino) )-2-hydroxy 7°lovansulfonic acid, piperazine-N, N-nohis (2-ethanesulfonic acid), piperazine-N, N'-bis(2-hydroxypropanesulfone fi), N-)lis(hydroxymethyl) Methyl-3-aminopropanesulfonic acid, N-tris(hydroxymethyl)methyl-2
-Hydroxy-3-aminopropanesulfonic acid, N-)
Examples include tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and tris(hydroxymethyl)methylglycine. Furthermore, compounds containing carboxylic acids and sulfonic acids can also be used.

These are used in the form of salts, and for example, they can be used as buffering agents, but when used not as buffering agents, they may be used in amounts of about lθ% to about It is preferable to add 250%.

In the case of immunohistochemical staining, a direct antibody method in which an enzyme is directly attached to an antibody or an indirect antibody method in which an enzyme is labeled to a secondary antibody is used. The labeling enzyme used in this case is preferably peroxidase, alkaline 7-osphatase, β-D-galactosidase, or the like. Labeling of an enzyme with an antibody can be carried out by a known method, for example, the method described in "Enzyme Immunoassay J" edited by Eiji Ishikawa et al. (1978, Igaku Shoin Series).

When using peroxidase, Kazuhisa T
Written by aketaa rJ, 1mtruno1. Method
alkaline phosphatase, β-D-
Regarding galactatosidase, the tetrazolium salt of the present invention can be colored by using an electron transfer agent-releasing substrate.

The electron transfer agent-releasing substrate refers to a substrate for alkaline 7-osphatase or β-0-galactatosidase that is hydrolyzed by the enzyme and releases an electron transfer agent for reducing the tetrazolium salt. Examples of the electron transfer agent-releasing substrate include ifhindryl derivatives such as 5-bromo-4-chloro-3-indolyl-phosphoric acid and 5-bromo-4-chloro-3-indolyl-β-D-galactoside.

These reagents can be used directly on tissues, such as immunohistological staining, Western blot, Southern blot, and dot plot methods.
Alternatively, it can be preferably used for staining on a membrane.

On the other hand, as mentioned above, the reagent composition of the present invention can also be usefully used in multilayer analytical elements.

The tetrazolium salt according to the present invention can be contained in the reagent layer (i.e., the detection layer) of the multilayer analytical element, and the specific anion species according to the present invention can be contained in the same layer and/or other layers. .

One embodiment of the use of the multilayer analytical element of the present invention is the measurement of reduced coenzyme in the presence of an electron transfer agent. These are JP-A-59-44658, JP-A-59-91896, JP-A-63-52897, JP-A-63-71199, and JP-A-1989.
-320999 etc. can also be used. On this occasion,
Examples of the electron transfer agent include 1-methoxy 7-enazine methosulfate, phenazine methosulfate, and meltable diaphorase, but diaphorase is preferable. In this embodiment, dehydrogenases such as lactate dehydrogenase can be measured, and dehydrogenases can be further contained in the multilayer analysis element to measure glycerin, triglyceride, GOT, GPT, etc.
, creatinine kinase, and other enzyme activities can be measured.

Yet another embodiment is a combination with an electron transfer agent-releasing substrate. As described above, this substrate uses an indolyl derivative, and includes a condensate of the derivative with a phosphoric acid ester and β-D-galactose. These include, for example, JP-A No. 59-88097, JP-A No. 5991896 and JP-A No. 6
It is easily prepared according to the method described in No. 1-262660 and No. 64-71499.

Moreover, as another aspect, the above-mentioned Kazuhisa Tak
eta et al. rJ, Immunol, MethodJ 1
According to the method described in Volume 986.95, pages 71 to 77,
By incorporating it into a multilayer analytical element, it can be applied to peroxidase-based analytical reactions.

〔Example〕

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

Example 1 (Immunohistological staining) A pancreatic section was stretched on a slide glass and stained with anhydrous acetone for 5 minutes.
After fixation for ~10 min, 5% normal goat serum was used for 10 min.
A masking operation was performed for ~20 minutes to suppress non-specific immune reactions.

After reacting this sample with an alkaline 7-osphasase-labeled anti-glucagon antibody prepared according to a conventional method, the sample was reacted with a phosphate buffer (0,
It was washed with 1M-PBS) and used as a sample.

Next, a reaction solution was prepared as follows.

Substrate solution 5-bromo-4-chloro-3-0,24 Indolyl phosphate (toluidinium salt) Magnesium chloride 5 N Anionic species of the present invention (shown in Table 1) 100mM Tris-HCl buffer 100mff after dissolution pH-9,
Adjusted to 5.

Color developer tetrazolium salt (shown in Table 2) Anionic species of the present invention (shown in Table 1) Magnesium chloride 100mM - Tris-HCl buffer 100m
ff After dissolution, adjust the pH to -9.5.

Table-2 C! H.

OHC*Hs The samples are compounds A-D in Table 2 and N in Table 1.
A combination of O1 is displayed as A-1, . . . D-6, etc.

Equal amounts of the substrate solution and coloring solution were mixed, dropped onto a slide glass on which the sample was placed, reacted for 30 minutes at room temperature, and washed several times with PBs.

Furthermore, in accordance with a conventional method, after fixation with a 20% formalin solution, control staining was performed using methyl green.

This was dehydrated with an ethanol column, cleared with xylene, and then sealed with epoxy resin.

After the xylene of the epoxy resin was completely evaporated, optical microscopic observation was performed. The results are C-1 to 6 and D-1 to 6.
In this case, the formazan dye developed during the dehydration process or clearing process was eluted, and a sufficient concentration could not be obtained.

On the other hand, A-1 to 5 and B-1 to 5 were stained red and blue-black at sufficient concentrations at glucagon localization sites, and the contrast with the methyl green staining was also good.

In addition, some elution of the dye was also observed for A-6 and B-5.

Example 2 A phosphate buffer solution (hereinafter P
Serially diluted lμQ of goat IgG was spotted using BS (referred to as BS).

After air drying, blocking was performed overnight at 4°C with a 1% bovine serum algomine (BSA)-PBS solution, and then alkaline 7-osphatase-labeled rabbit anti-goat IgG antibody (manufactured by Kappel; diluted 1500 times with a 1% BSA-PBS solution) 2 hours at 4°C. 0.05%
It was washed five times with a Tween-20 (polyoxyethylene sorbitan monolaurate, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)-PBS solution, and immersed in a coloring substrate solution. The substrate solution for color development is the following 2
It is a kind.

(1) 5-bromo-4-chloro-3-indolylphosphoric acid
0.12mM magnesium chloride
5-nitro TB-OA (compound of the invention)
0.5+mMNN-2-hydroxyethylpiperazine-N'-21,0-M-ethanesulfonic acid sodium chloride 20
mM trishydroxymethylaminomethane
5 mM was dissolved in pure water, the pH was adjusted to 8.5, and the final volume was 100 ml.

(2) In place of the above compound of the present invention, nitro-TB 0.5-
The same product was prepared except that M was dissolved in 0.5 mff1 of dimethylformamide and mixed.

After reacting for 15 minutes, it was thoroughly washed with water and air-dried. When using the coloring substrate solution (1), 0.05 ng of goat IgG was used for detection, but with the comparative substrate solution (2), 1 ng of goat IgG was used for detection.
Only up to the spot was detected.

Example-3 As in Example-2, goat IgG was applied to the nitrocellulose membrane.
A plot of serially diluted samples was prepared. Similarly, at 4°C with 1% BSA-PBS solution.

After blocking overnight, peroxidase-labeled rabbit anti-goat 1gc antibody (manufactured by Kappel; 1% BSA-
Dilute 1500 times with PBS solution) and 4°C.

The reaction was allowed to proceed for 2 hours. Then 0.05% Tween-20
(Polyoxyethylene sorbitan monolaurate: manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) - Washed five times with a PBS solution and immersed in a coloring substrate solution.

(1) Phenol 0.5-TB
-OA 0.5mM NADH 0.7mM 30%H! 0. 2
0 ng Triton

Biotinylated DNA was prepared according to a conventional method for complementary λ phage DNA (sectioned with Hind m), and (1)
The nitrocellulose filter prepared above was placed in a heat seal bag, prehybritized at 42°C for 2 hours, biotinylated DNA was added, and hybridization was carried out for 16 hours.

Further, the nitrocellulose filter was washed three times with distilled water, reacted with a streptavidinated alkaline 7-ostase solution at room temperature for 2 hours, and then washed three times with distilled water.

As a substrate solution for color development, it was immersed in (1) and (2) shown in Example-2 for 12 hours at room temperature to react.

As a result, color development was confirmed up to 0.5 pg spot in case (1), but up to 5 pg spot in case (2).

(2) The nitro-TB-OA of (1) above is converted into nitro-T
It was the same except that it was changed to H.

After reacting for 15 minutes at room temperature, it was thoroughly washed with water and air-dried.

The color-developing peribasal fluid (1) has a concentration of 0. Although spots were confirmed up to 5 ng of goat IgG in (2), 5 ng of goat IgG was detected.
I was only able to confirm up to G.

Example 4 (1) Dot blotting on a nitrocellulose filter Lambda phage DNA heat-treated into a single strand on a 2×3c nitrocellulose filter (cleaved with Hind m)
0. ipgs O, 5pgs 1 pgs 5
PgN 7.5pg%10f'g1100pgslng
and lOng were spotted to a diameter of 4+++ orchards and baked at 80° C. for 2 hours under reduced pressure.

(2) Creation of DNA globe 7 Otobiotin labeling end Deteeximin kit (Photobiotin Labellin)
g and Detection Kit) (BRES
A biotin-labeled DNA probe was prepared using equal amounts of A Pty, Ltd. (obtained from Nirapon Gene Co., Ltd.) and the λ phage DNA (cleaved with Hind■) according to a conventional method.

(3) Hybridization The nitrocellulose filter prepared in (1) was placed in a heat-sealed bag and prehybridized at 42°C for 2 hours, then the DNA probe from (2) was added, and the
Time hybridization was performed. In addition, hybridization is T, Majyatiset al rM
Olecular Cloning A Labora
tory ManualJCold Spring H
Arber Laboratory 1982 was followed.

This filter was washed with water three times.

(4) Place the color reaction filter in a heat-sealed bag and add 3% bovine serum albumin solution (NaCQ, 2mM MgCQ, 0.0
1-0M 100m containing 5% TritonX-100
M) After adding bovine serum albumin dissolved in Lis-HCl buffer (pH = 7, 5) and incubating at 42°C for 20 minutes, the BSA solution was discarded and the above buffer was labeled with alkaline phosphatase. Add biotin and incubate for 10 minutes at room temperature. Take out the filter from the bag, wash it twice with the above buffer solution, and then
mM Tris-HCl buffer (pH=9.5100mMNa
cffi (contained in 5mM MgCQ, ') twice, and washed with substrate solutions (1) and (2) of Example-2 for 5 hours.
Carry out the color reaction at room temperature.

The results were 0.0 for substrate solution (1), which is the substrate of the present invention. l
Spots could be observed up to pg, but in case of (2) it was up to 5 pg.

Example 5 (Analytical element for GOT) The following first reagent layer (R-1) was provided on a transparent undercoated polyethylene terephthalate support with a film thickness of 180 μm.

First reagent layer (R-1) Gelatin glutamate dehydrogenase diaphorase adenosine-5'-diphosphate-potassium salt nitro-TB-
OA Triton Trishydroxymethylaminomethane glutamic acid dehydrogenation oxygen adenosine-5'-diphosphoric acid potassium salt Nobiscol V
A-28* Triton
0: 80) 5.70 (g/■su1.22 (g/rice) /s 42.000 (IJ/■su 2.100 (■/■9 1.8 0.78 (g/■ spread layer (S) powder filter paper [Toyo Roshi Co., Ltd. 40-100 mesh]
91 (g/-〇L-Sodium Aspartate 0.39 (g/+*Riketoglutar 0.50
(g/m oxidized nicotinamide adenine dinucleotide 5.5 (g/m”) glutamate dehydrogenase - (U/m rearenosine-5'
-diphosphoric acid-potassium salt 2.3 (g/m
”) Styrene-glycidyl methacrylate 23 (g/m copolymer (weight ratio 9:l) Triton
Instead, a product was prepared in which 1.2 g/m'' of nitro-TB was substituted.

The former was used as an analytical element of the present invention, and the latter was used as a comparative analytical element.

For the analytical element of the present invention and comparative analytical element, Konica Dry Lab "80M" (manufactured by Konica Corporation) was used.
-U to 8, -U to 125 and -U to 233 (7) GO
After spotting 10 μα of human serum with T activity, 37%
After incubation, the reflection density was measured after 3.5 minutes and 7 minutes using a 546 nm filter.
The difference in reflection density was determined.

GOT activity 28 125 233 Analytical element of the present invention 0.045 0.189 0.29
3 comparative analysis elements 0.011 0.093 0.1
As shown in the results above, it can be seen that the analytical element of the present invention has better sensitivity than the comparative analytical element.

Furthermore, when the developing layer of the analytical element was observed after the measurement was completed, it was found that the comparative analytical element was stained blue, the generated dye had migrated to the developing layer, and coloring was highly uneven on the transparent base side. None of these were observed in the analytical element.

Claims (2)

[Claims] (1) A biological analysis reagent composition comprising a compound represented by the following general formula [I] and/or a compound represented by the general formula [II] and a specific anion species. General formula [I] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ General formula [II] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, Y is a linear alkylene group with 2 to 4 carbon atoms, It may have a substituent, and one hydroxyl group may be present as the substituent. R^1 is an alkyl group having 1 to 4 carbon atoms or a hydroxyethyl group, R^2 is an alkyl group having 1 to 4 carbon atoms,
It may have 1 to 2 hydroxyl groups or one phenyl group. W is a hydrogen atom or a nitro group. Z is a 4,5-dimethyl-2-thiazolyl group when W is a hydrogen atom, and a 4-iodophenyl group when W is a nitro group. R^3 is a methoxy group, but when W is a hydrogen atom, it may be a hydrogen atom. X^■ is a counter ion that cancels out the charge in the compound,
It is a chloride ion or a bromine ion. There are also mathematical formulas, chemical formulas, tables, etc. The substitution position in the benzene ring is the 3rd or 4th position. ] (2) A compound represented by the above general formula [I] and/or a multilayer analytical element constructed by sequentially laminating a layer containing at least a hydrophilic polymer and a porous development layer on a light-transmitting support.
Or a multilayer analytical element characterized by containing a compound represented by the general formula [II] and a specific anion species.