JPH01308967A - Immunological analysis and analysis element therefor - Google Patents

Abstract

PURPOSE:To enable a low-noise analysis with a higher sensitivity by arranging a substance, which is specifically bonded to a label substance in a specified analysis system to modulate a signal attributed to the labelling substance, as aryl mercury derivative to further contain a compound as given by a specified formula and/or a salt thereof. CONSTITUTION:In a method of analyzing a specified component in a fluid sample by a specified analysis system, a substance which is specifically bonded to a label substance a used and modulates a signal attributed to the labelling substance is an aryl mercury derivative and further contains a compound as given by the formula (wherein R1, R2, R3, R4 and R5 represents C1-5 alkyl group having an hydroxide group or sulfornic groups as substitution group in a main chain and a salt thereof. Thus, the aryl mercury derivative maintains a enzyme blocking capacity at a high level and a low noise analysis can be realized with a high sensitivity using buffers in which a labelling enzyme displays a high activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for analyzing trace components in a fluid sample and an analytical element used therefor, and particularly to analysis of specific trace components in biological fluid samples. The present invention relates to a method and an analytical element thereof.

[Conventional technology]

Various analytical methods have been developed to detect trace amounts of substances contained in biological fluid samples.

The analytical 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.

The immunoassay method was developed in 1958 by Berson.
Radioimmunoassay has been widely used since John and Yallow succeeded in measuring in/nilin in serum using radioactive iodine-labeled raninsulin and anti-insulin antibodies in the serum of diabetic patients. It is being

Since then, various labeling compounds other than radioactive isotopes have been developed, such as enzymes,
Enzyme substrates, coenzymes, enzyme inhibitors, batatteriophages, cycling reactants, metal and organometallic complexes, organic prosthetic groups, chemiluminescent reactants, fluorescent molecules, and the like.

One of the important technical problems regarding the above immunoassay is the separation of substances that have bound (hereinafter abbreviated as B) and substances that have not bound (hereinafter abbreviated as F) (hereinafter referred to as B/
(abbreviated as F separation).

Various methods have been developed and proposed to solve the problems in the immunoassay (for example, Japanese Patent Application Laid-Open No. 53-3
No. 8619, No. 53-79024, No. 55-9085
No. 9, No. 57-67860, No. 57-200862, No. 58-18167, No. 59-77356 and No. 59170768).

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

On the other hand, in wet chemistry, an immunoassay method using a competitive method using a stationary phase has been developed (for example, JP-A-58-209994 and JP-A-59-202064).
.

However, in these measurement methods, competitive reactions are performed between two substances fixed on a carrier in a separate stationary phase in a relatively large amount of aqueous solution, and the substance in the solution, so that the intended binding may not occur. However, since the overall enzyme activity is measured without distinguishing between the two stationary phases, the results may not be satisfactory in terms of sensitivity, accuracy, and reproducibility due to background and noise problems. I can't get it.

On the other hand, in dry chemistry, an immunoassay method using a second antibody has been developed (Japanese Patent Laid-Open No. 57-82776).
No. 57-82767).

However, there is room for improvement in these methods, such as the complicated operations and the need for techniques for interlayering with good reproducibility. Further, Japanese Patent Application Laid-Open No. 59-34155 discloses a method using an unbound material storage sheet. However, even with this method, the above-mentioned problem will occur if the reaction sheet and the unbound substance storage sheet are kept in close contact with each other, and it is cumbersome to separate the two sheets during measurement, especially if the measurement is automated. It becomes an obstacle when doing so.

As a means to solve these conventional B/F separation problems, a method has been recently invented that uses a substance that specifically binds to an unreacted labeling substance and modulates the signal caused by the labeling substance.

This technology is disclosed in Japanese Patent Application Laid-Open Nos. 61-292060 and 62-90.
No. 539, No. 62-90538, No. 62-24725
No. 6, No. 62-249063, No. 62-249060
No. 62-267666, No. 62-267667, No. 63-45562, etc.

Generally speaking, these techniques are methods for measuring and analyzing a specific component in a fluid sample using a binding reaction with a substance that can specifically bind to the specific component, and at least the analysis system includes: In the analysis process for a specific component in a fluid sample, a substance that specifically binds to the specific component is bound to either a biologically active substance that does not bind to the specific component or a substance that specifically binds to the biologically active substance; Either a label formed by binding a labeling substance to the specific component or its analogue, or a label formed by binding a labeling substance to a substance that specifically binds to the specific component, and C1 at least one type of carrier. Either a substance that specifically binds to the biologically active substance or a biologically active substance that does not bind to the pre-grasping identification component, and a substance that specifically binds to the label/label substance and modulates the signal caused by the label substance (specific modulator substance). ) and are separately immobilized on the same or separate carriers and are comprised of a combination of porous reaction layers.

As a result of detailed studies on these techniques, the present inventors used a labeled enzyme that is inhibited by arylmercury as the labeling substance, and used a specific modulating substance (for example, a color light-absorbing substance).
It has been found that particularly sensitive measurements can be performed when an arylmercury derivative is used as the labeling enzyme, and more preferably when β-D-galactosidase is used as the labeling enzyme.

An essential requirement for measurements in each of the above analytical systems is that the enzyme activity of a substance that specifically binds to a specific component or a labeled enzyme that is not immobilized on a carrier can be efficiently inhibited by arylmercury immobilized on a carrier. be.

Therefore, it is clear that the buffer contained in the reaction solution of the above-mentioned analytical system must not have the effect of reducing the inhibitory effect of arylmercury.

Furthermore, since the measurement is performed based on the enzymatic activity of a labeled enzyme bound to a substance that specifically binds to a specific component or a carrier, it is clear that the buffer must not inhibit the enzymatic activity.

[Problem to be solved by the invention]

However, upon further investigation, it was discovered that it was extremely difficult to select a buffer solution in the above-mentioned analysis system.

That is, for the purpose of retaining enzyme activity, buffers commonly used by those skilled in the art include phosphates, tris(hydroxymethyl)aminomethane, glycine, citric acid, and the like.

However, when these buffers are applied to the aforementioned assays,
It was discovered that the background enzyme activity was relatively high and was an obstacle to increasing the sensitivity of the analysis.

Further investigation into the cause revealed that the presence of these buffers significantly reduced the enzyme inhibiting ability of the arylmercury derivatives immobilized on the carrier.

Therefore, an object of the present invention is to provide a highly sensitive and low-noise analytical method using a buffer in which an arylmercury derivative maintains a high level of enzyme inhibitory ability and a labeled enzyme exhibits high activity, and an analytical element implementing the method. Our goal is to provide the following.

[Means to solve the problem]

The object of the present invention is to analyze a specific component in a fluid sample by: (a) the specific component bound to either a biologically active substance that does not bind to the specific component or a substance that specifically binds to the biologically active substance; a substance that specifically binds to; b. a label formed by binding a labeling substance to the specific component or its analogue;
or a labeled body formed by binding a labeling substance to a substance that specifically binds to the specific component, and c. At least one carrier containing a substance that specifically binds to the biologically active substance or an organism that does not bind to the specific component. Formed by a combination of a porous reaction layer containing either one of the active substances and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance, separately immobilized on the same or separate carriers. or (a) a labeled body formed by binding a labeling substance to the specific component or its analog, or a labeled body formed by binding a labeling substance to a substance that specifically binds to the specific component; b. At least one carrier contains a substance that specifically binds to the specific component and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance, separately immobilized on the same or separate carriers; In the method of analyzing the specific component by selecting one of the analysis systems using a combination of a porous reaction layer and a porous reaction layer, the substance that specifically binds to the labeling substance and modulating the signal caused by the labeling substance is an arylmercury derivative. Furthermore, compounds represented by the following general formulas (r) and (I[) and/or
or a salt thereof, and an immunological analysis element according to the method.

General formula (1) In the formula, R, , R, , R, , R, and R6 represent an alkyl group having 1 to 5 carbon atoms and having a hydroxyl group or a sulfone group as a substituent in the main chain. More preferably, the compound represented by the general formula [I] is bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.

General Formula ([1) In the formula, R1 and R2 represent an alkyl group having 1 to 5 carbon atoms and having a hydroxyl group or a sulfone group as a substituent in the main chain. More preferably, the compound represented by the general formula [■] is piperazine-N, N'-bis(2-ethanesulfonic acid) or N
-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid is preferred.

Incidentally, the compounds represented by the general formulas (1) and (n) may be used in combination.

Specific examples of the compound represented by the general formula [I] used in the present invention are shown below, but the invention is not limited thereto.

1-1゜(HOCHz:hCN(CHzCHzOH)xl-2, CH2C)+201(I-3, (HOC)IJ*CN(CHzCHzSOJ)zl-4
, T-5, (HOCH*CHi)scN(CHzOH)zI-6, 1-7, 1-8, OH0H 1-9, -10, CH,OH These compounds are in the Archives of Bioch
emistryand Biophysics vol. 96 p653 (1962) and Analytica
l Biochemistry Volume 104 p300
(1980).

In particular, the embodiment in which R, , R, , R are all CH20H is a preferred embodiment because synthesis is easy using tris(hydroxymethyl)aminomethane, which is often used in the art, as a starting material.

Depending on the acid dissociation constant (pKa) of these compounds, a part of them is converted into a salt using a strong base such as sodium hydroxide or potassium hydroxide or a strong acid such as hydrochloric acid, and the pKa of the solution is
It is preferable to adjust the labeling enzyme using H to the optimum level 1) H.

Furthermore, it is advantageous to consider the optimum pH of the labeling enzyme used and use a compound having an acid dissociation constant (pKa) as close to the optimum pH as possible.

For example, when using β-D-galactosidase derived from Escherichia coli as a labeling enzyme, bis(2-and droxyethyl)iminotris(hydroxymethyl)methane of Compound Example 1-1 is selected and a portion thereof is neutralized with hydrochloric acid. By p
A preferred example is to use H in the range of 6.5 to 8.5, more preferably 7.0 to 7.5.

Next, specific examples of the compound represented by the general formula [II] used in the present invention are shown below, but are limited to n-1, l-λ 1-10,5CH2CH2N NCH2CH,S
o, H-y I[-2, 11-3, υn ■ -4, n-5, ff-6, It-7, 11-8, 11-9, ll-10, These compounds are available in Archives or Bioch
Emistryand Biophysics Vol. 96 p653 (1962) and Analyticl
BiochaIllistry Volume 104 p300
(1980).

Depending on the acid dissociation constant (pKa) of these compounds, a part of them is converted into a salt using a strong base such as sodium hydroxide or potassium hydroxide or a strong acid such as hydrochloric acid, and the pKa of the solution is
It is preferable to adjust the pH to the optimum value for the labeling enzyme using H.

Furthermore, it is advantageous to consider the optimum pH of the labeling enzyme used and to use a compound having an acid dissociation constant (pea) as close to the optimum pH as possible.

For example, when using β-D-galactonase derived from Escherichia coli as a labeling enzyme, piperazine=N,N'-bis(2-
ethanesulfonic acid) or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, partially neutralized with hydrochloric acid to give a pu of 6.5 to 8.5, more preferably 7.0. As a preferable example, it is used within the range of 7.5 to 7.5.

The concentration of these compounds varies depending on the type of specimen or the type of analysis system, but is generally between 0.001 and IOM at the time of measurement.
, more preferably in an amount ranging from 0.01 to 1M.

Furthermore, it is often more preferable to allow Mg2+ ions to coexist in 0.1 to 100+IIM during measurement.

Methods for applying the above-mentioned compounds include a method in which the compound solution is mixed with a fluid sample in advance and then dropped onto the analytical element, and a method in which the compound solution is incorporated in the analytical element in advance.

In both cases, JP-A-61-292060 and JP-A-62
-90539, 62-90538, 62-24
No. 9063, No. 62-267667, Patent Application No. 1986-2
This can be easily achieved by using the buffering agent application method described in No. 80166 as is.

In the present invention, any form of solution or colloidal solution can be used as the fluid sample, but fluids of biological origin are preferably used, such as blood, plasma, serum, brain, spinal fluid, saliva, amniotic fluid, milk, etc. Examples include urine, sweat, meat juice, etc.

The specific components in a fluid sample that can be measured by the present invention are:
A substance or group of substances whose presence or amount in a fluid sample can be determined to obtain a substance that specifically binds to that particular component. That is, polypeptides, proteins, complex proteins,
Examples include polysaccharides, lipids, complex lipids, nucleic acids, hormones, vitamins, drugs, antibiotics, agricultural chemicals, and the like. Specifically, the following substances or substance groups can be mentioned;
It is not limited to these.

Table 1 (Proteins, complex proteins) Prealbumin, albumin, α1-acid glycoprotein, σ
1-antitrypsin, GL-glycoprotein, transflutin, α,-antichymotrypsin, α1-lipoprotein, thyroxine-binding globulin, ceruloplasmin, Z
n-α2-glycoprotein, Gc-globulin, interα-
Trizcin isohibita, α1-macroglobulin, α,
-FIS-glycoprotein, (12-macroglobulin, haptoglobin, α, -lipoprotein, hemovexin, transferrin, β-lipoprotein, β□-N protein, β2-
macroglobulin, C-reactive protein, myoglobin,
Erythromycin, immunoglobulin (IgG, IgM
, IgA, IgD, IgE), complement system components (C+
q, clr, cls, c2. c3. c4.
c@, c@, C7+CM+ cs, etc.), fibrinogen, hemoglobin, glycated hemoglobin, blood coagulation factor, 1 (Bs anti-W, HBs antibody, enzymes (e.g., acid phosphatase, alkaline phosphatase, alkaline phosphatase isoenzyme, σ-amylase,
amylase isoenzyme, aldolase, cholinesterase, creatine phosphokinase, creatine phosphokinase isoenzyme, transaminase (
GOT, GPT), lactate dehydrogenase, lactate dehydrogenase isoenzyme, γ-GTP, lipase, monoamine oxidase, leucine aminopeptidase, glucose-6-phosphate dehydrogenase, etc.), etc. (hormones and hormone-like substances) Follicle stimulation hormone (FSH), structure-stimulating hormone (LH)
), growth hormone ((J), thyroid stimulating hormone (TS)
R), adrenocorticotropic hormone (ACTH), melanin-stimulating hormone (MSH), vanprecin, oxytocin, insulin, glucagon, angiotenin I and ■, prolactin, secretin, dopamine, serotonin, somatostatin, thyroxine (Tt), tri Iodothyronine (T,), gastrin, flutisol, aldosterone, catecholamines, estrogen,
Glogesterone, testosterone, placental gonadotropins, placental lactogen, pituitary hormone releasing factor (T
RH, FSH-RH.

CRH, LH-R11, etc.) etc. (vitamins) Biotin, thiamin, vitamin A1, vitamin B3, vitamin B12, vitamin C1, vitamin D1, vitamin E
1 Vitamins and 1 folic acid, etc. (It @ tumor marker) α-fetoprotein, carcinoembryonic antigen, ferritin, polyamine, visceral carcinoembryonic antigen, basic fetoprotein, M
-Protein, prostatic acid phosphatase, carbohydrate antigen (CA
19-9. CA125, etc.), Garigliosize (various drugs and metabolites) Bensoylecgonine, cocaine, codeine, dextrometrophan, heroin, lysergic acid, morphine, quinidine, quinine, amikacin, gentamicin, kanamycin, neomycin, tobramycin, actinomycetin, chromomycin, chloramphenicol, chlormycetin, chlortetracycline, erythromycin, oxytetracycline, penicillin, polymyxin 81 terramycin, tetracycline, streptomycin, diphenylhydantoin, ethosuximide,
Phenobarbital, brimidone secobarbital, acetaminophen, amitributyrin, carbamazepine,
Digoxin, ginviramide, lidocaine, mentrexate, N-acetylprocainamide, phenytoin, procainamide, propranolol, theophylline, canapinol, tetrahydrocanapinol, cholinergics, antihistamines, atrobin, butyrophenone, caffeine, chlorpromazine, epinephrine , griseofulvin, imipramine, L-dopa, meperidine, mesolobamate, methatone, narcein, nortributyrin, oxazepam, bapaverine, prostaglandin, tegretol, valproic acid, etc. and their metabolites (microbial surface markers), bacterial antigens, fungal antigens, Parasitic antigens, viral antigens (pesticides), halogenated biphenyls, phosphate esters, thiophosphates, and their metabolites (others), blood type substances, calciolibin, allergens, etc. Also, specific components in the fluid sample in the present invention An analogue of means a substance that can competitively bind to the specific component with respect to a substance that can specifically bind to the specific component, which will be described later.

The arylmercury derivative used in the present invention is an organic mercury compound represented by the general formula R(HgX)n.

In the general formula, R represents benzene or a 7talene ring, and may have a substituent, such as a halogen atom (F, CQ, Br, I), -CN.

Examples include an alkyl group and a phenyl group that may have a substituent, and RI and Rl represent an alkyl group and a phenyl group that may have a substituent and a hydrogen atom.

The number of substituents on the benzene or 7talene ring is 7 or less, but preferably 3 or less. X represents a monovalent acid group.

n represents an integer of l or 2.

Specific examples of the arylmercury derivative in the present invention are shown below, but the invention is not limited thereto.

CH.

For other arylmercury derivatives, see Beilsteins Handbuch 7 der Organissien Hemi-
Organischen Che+5ie) (4th edition) Volume XrV, 2nd Supplement, pp 1347-1424, and these derivatives can be synthesized according to the methods described above.

Arylmercury derivatives react specifically and with high affinity with thiol groups. On the other hand, many enzymes have a thiol group in their molecular structure, and particularly many have a thiol group at a site involved in activity. Such enzymes bind to the above-mentioned arylmercury derivatives and undergo a change in their activity [see "Enzyme Ndobusoku" (edited by Bunji Maruo and Nobuo Tamiya, published by Shokusen Shoten, 1982), etc.].

Therefore, by using an arylmercury derivative as an inhibitor of the free labeling enzyme and an enzyme having a thiol group in the site involved in such activity in the labeling enzyme, it is possible to specifically bind to a specific component. The labeled enzyme that did not react directly or indirectly with the substance can be trapped by arylmercury specifically and with strong affinity, and the activity of the labeled enzyme can be changed, so the reaction system It is possible to actively perform B/F separation within. Furthermore, as mentioned above, there are many enzymes that have a thiol group in the site involved in their activity, so it is possible to select a labeled enzyme from a fairly wide range.Once a system using an arylmercury derivative is established, Even if it is necessary to change the labeled enzyme due to the addition of a measurement target, as long as the changed labeled enzyme undergoes a change in activity by the arylmercury derivative, it can be immediately applied to this system. This will be very advantageous for expansion.

Substances selected from the group consisting of specific components, analogs of specific components, and substances that specifically bind to specific components (hereinafter referred to as "substance E")
A labeled substance in which a substance E and a substance that specifically binds to a specific component or a specific component is bound, and a labeled substance that retains the specific binding ability between the substance E and a substance that specifically binds to a specific component or a specific component, and which binds the above-mentioned labeled enzyme. This is a general term for substances in which the substance E and the enzyme are bound directly or indirectly by chemical means while retaining the ability to emit signals. In fact, it can be obtained by combining the substance E and the enzyme with a known reagent and a known method well known to those skilled in the art. "Enzyme immunoassay (2nd edition)" (Igaku Shoin, published in 1978) and "Clinical Pathology" in Japanese Clinical Pathology Kinpei.
Extra special issue No. 53 "Immunoassay for clinical testing - technology and applications" (Clinical Pathology Publishing Association, 1983)
You can refer to the methods described in, etc. In order to help the understanding of the present invention, specific examples will be described below, but the present invention is not limited thereto.

(1) Method of reacting the substance E and the enzyme with a crosslinking agent such as the following ■ 2,4.6-) Lichloro-1.3.5-triazine ■ 4.4'-difluoro-3,3'-dinitro Diphenylsulfone ■ Toluene-2,4-diisocyanate ■ N, N'
-Dicyclohexylcarbodiimide ■ l-(3-dimethylaminopropyl)-3-ethylcarbodiimide ■ Bisdiazo-dianisidine ■ Glutaraldehyde etc. (2) At least one of the substance E and the enzyme is 1
1i chain, a method in which the sugar chain is treated with periodic acid and the resulting aldehyde group is reacted with the amino group of the partner substance to be bonded.

(If necessary, the substance E or the enzyme should be pretreated with 1-fluoro-2,4-dinitrobenzene or the like to prevent the formation of unnecessary bonds during periodic acid treatment. Treatments such as controlling the pH of the acid treatment reaction to 4 to 5, or stabilizing the bond formed by the book base between the substance E and the enzyme with sodium borohydride, ethanolamine, etc. may be performed. (3) If the substance E and the enzyme have a thiol group, or can generate a thiol group by reduction etc., or can introduce a thiol group by treatment with an appropriate compound, it is known as a maleimide reagent. A method of reacting the thiol group with various crosslinking agents.

(Here, the following examples are given as compounds into which a thiol group is introduced. ■ S-acetylmercaptosuccinic anhydride ■ Methyl-3-mercaptopropionimidate ■ Methyl-4
-Mercaptobutyrimidate■ 2-iminothiolane■ 3-(2'-dithiopyridyl)propionic acid N-hydroxysuccinimide ester■ Methyl 3-(4'-dithiopyridyl)propionic acid and the aforementioned maleimide Examples of reagents include:

■ NIN'-0-phenylene dimaleimide ■ N, N
'-P-phenylene dimaleimide■ N,N'-m-phenylene dimaleimide■ N,N'-oxydimethylene cimaleimide■ N,N'-succinimidyl-N-maleimide acetate■ N-succinimidyl-4-(N -maleimido)butyrate■ N-succinimidyl-5-(N-maleimido)heptanoate■ N-succinimidyl-6-(N-maleimido)hexanoate■ N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [ Phase] N
-Succinimidyl 1-(N-maleimido)benzoate ■ N-succinimidyl-p-(N-maleimidophenyl)-4-butyrate @ N-sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate ON-sulfosuccinimidyl-a-(N-maleimido)benzoate o N-sulfosuccinimidyl-p-(N-maleimidophenyl)-4-butyrate @ N-succinimidyl-4-(N-maleimidomethyl) Benzene-1-rupoxylate, etc.) (4) A method of introducing a pyridyl disulfide group into the substance E or the enzyme and reacting it with a thiol group that has been introduced into the partner compound to be bound or is originally present.

[The pyridyl disulfide group may be introduced by treatment with 3-(2'-dithiopyridyl)propionic acid N-hydroxysuccinimide ester, methyl-3-(4'-dithiopyridyl)propionimidate, or the like. The introduction of a thiol group can be carried out using the methods described in (3). If a thiol group can be introduced by treatment with a compound, the thiol group of one of the substances is converted into a pyridyl disulfide group, and the thiol group of the other substance is reacted with the thiol group of the other substance to be bonded.

(Conversion of thiol group to pyridyl disulfide group is
This can be carried out using 4.4'-dithiodipyridine or the like. ) (6) present or introduced into the substance E and the enzyme;
A method of reacting an amino group or a thiol group with p-benzoquinoline.

(7) A method of reacting monoiodoacetic acid N-hydroxysuccinimide ester with a thiol group present or introduced into the substance E and the enzyme.

(However, in the methods (3) to (7), the bonding between the substance E and the enzyme is performed under conditions such that the thiol group involved in the activity present in the enzyme does not participate in the bonding with the substance E. (8) A method of reacting an antibody against the substance E, an antibody against the enzyme, and an antibody that specifically binds to the former two antibodies.

(9) Substance E1 A method in which one of the enzymes is bound to avidin and the other to biotin, and both are bound by biotin-avidin binding.

Labeled enzymes that can be applied to the present invention are enzymes that bind to arylmercury derivatives and change the enzyme activity, and representative examples thereof are listed in Table 2. The activities of these enzymes can be measured using activity measurement methods suitable for each enzyme.

Table 2 1%l, l, 1 alcohol dehydrogenase 1
, L 1,2 alcohol dehydrogenase (NA
DP 1.1% L, 8 Glycerol-3-phosphate dehydrogenase 1% 1% 1,10 L-xylulose reductase 1
%111%18 myo-inositol-2-dehydrogenase 1.1%1,22 11DP glucose dehydrogenase LLI, 26 glyoxylate reductase 1%L
l, 27 Lactate dehydrogenase k1% 1129
Glycerate dehydrogenase 1% 1% 1,30 3-
Hydroxybutyrate dehydrogenase 1%111,37
Malate dehydrogenase 1% 1% 1140 Malate dehydrogenase (oxaloacetic acid carbonate XNADP
”) 1.1,1,41 Isocitrate dehydrogenase (
NAD 1.1,1,42 citrate dehydrogenase (NADP 1.1% l, 44 phosphogluconate dehydrogenase (decarboxylation) 1.1% 1,50 3a-hydroxysteroid dehydrogenase LLI, 95 phosphoglycerol dehydrogenase 111.3,■ Glycolate oxidase 1%1,3
,4 Glucose oxidase 1.2, Ll Formaldehyde dehydrogenase L2, I4 Aldehyde dehydrogenase (NADP") 112.1,5
Aldehyde dehydrogenase (NAD(P)'') I2.
1,12 Glyceraldehyde phosphate dehydrogenase 1.4.1.1 Alanine dehydrogenase 1.4.
1.3 Glutamate dehydrogenase (NAD(P)
) I4.1,4 Glutamate dehydrogenase (NA
DP) I4.3.3 D-amino acid oxidase 1.
5, I5 Methylenetetrahydrofolate dehydrogenase 1.9.6.1 Nitrate reductase (cytochrome) 1
．． 13.11.12 Lipoxygenase 114.13
．． 2 4-hydroxybenzoic acid 3-monooxygenase 1.14.16.1 Phenylalanine 4-° monooxygenase 2.1, 1.6 Catechol methyltransferase 2.1, 3.2 Asparagine carbamoyltransferase 2.1, 3. 3 Ornithine carbamoyltransferase 2.3.1,6 Choline acetyltransferase 2.3.1,7 Carnitine acetyltransferase 2,4,1,1 Phosphorylase 2,4,1,22 Lactose synthase 2,6,1
, 19 Aminobutyric acid aminotransferase 2, 7
, 1,1 hexokinase 2, 7, 1,2 glucokinase 2, 7, 1, I2 gluconokinase 2, 7, l,
19 Vocal sfoliprokinase 2, 7, 1132 Choline kinase 2, 7, 2, 1 Acetate kinase 2, 7, 2, 2 Carbamate kinase 2, 7, 3,
2 Taleatine Kinase 2, 7, 3, 3 Arginine Kinase 2, 7.4, 3 Adenylate Kinase 2,
7, 4, 6 Nucleoside diphosphate kinase 2, 7, 7
, 7 DNA nucleotidyl transferase 3, 1, 1, 3 1-lyacylglycerol lipase 3, 1% l, 13 cholesterol esterase 3
, 1,3,2 acid phosphatase 3,1,4,4 phospholipase D 3,1,31%l pancreatic endonuclease 3,2,1
, 1 α-Amylase 3, 2, 1, 2 β-Amylase 3, 2, 1114 Chitinase 3, 2, 1% 18 Neuraminidase 3.2.1, 2
0 a-D-glucosidase 3.2.1121 β
-D-glucosidase 3.2.1, 22 a-D-galactosidase 3.2.1123 β-D-galactosidase 3.2, ■, 26 β-D-fructofuranosidase 3.2.1, 31 β- D-glucuronidase 3.4.11.8 Pyroglutamyl aminopeptidase 3.4.14, l Dipeptidyl peptidase■
3.4.21%l Chymotrypsin 3.4.22.2 Papain 3.4.22.3 Ficin 3.4.22.4 Bromelain 3.4.22.6 Chymopapain 3.4.22.8 Clostri Pine 3.5.1,5
Urease 3.5.1,24 Choloylglycine hydrolase 3
．． 5.2.6 β-lactamase 3.5.3, l arginase 3.5.4.4 adonosine deaminase 3.6.1
11 Inorganic pyrophosphatase 3.6,
l, 9 Nucleotide pyrophosphatase 4, L 1
．． I pyruvate decarboxylase 4.1%11
39 Riprose diphosphate carboxylase 4.1,2
．． 11 Hydroxymanzolonitrile lyase 4.2
．． 124 Phosphobilinogen synthase 4.3.1
, 5 Phenylalanine ammonia lyase 4.3.
2.2 Adenylosuccinate lyase 5.1, 3.2
UDP glucose-4-epimerase 5.3.1,1
Triose phosphate inmerase 5.3.1, 6 Ribose phosphate inmerase 6.2, ■, 1 Acetyl-C
oA Synthetase 6.4.1,2 Acetyl-CoA
Among the above enzymes such as carboxylase, β-D-galactosidase can be mentioned as a particularly preferable example.

This will be explained below using β-D-galactosidase as an example.

β-D-galactosidase used in the present invention has enzyme numbers EC3, 2, and 1 as determined by the Enzyme Committee of the International Union of Biochemistry.
, 23, and those widely distributed in the animal, plant, and microbial worlds can be used. For example, E. coli, Pr
otaus Mirabilis, Saccharo
myes Fragilis, Ushigurumaru, Cat Liver, Pe
Examples include those purified from nicllium citrinum, but among these, those induced in Escherichia coli with lactose or the like are particularly preferred.

The activity of β-D-galactosidase can be determined, for example, by reacting a substrate as described below and measuring the change in absorbance or fluorescence intensity of the released aglycon.

(1) 5-bromo-4-chloro-3-indolyl-β
-D-galactoside (2) 5-bromo-2-naphthyl-β-D-galactoside (3) 5-carboxy-2-nitrophenyl-β-ri-galactoside (4) Isopropyl-β-D-thiogalactoside (5 )
4-methylumbelliferyl-β-D-galactoside (6) o-nitrophenyl-β-D-galactoside (7) o-nitrophenyl-1-thio β-D-galactoside (8) Phenyl-β-D- Galactoside (9)
3.6-hydroxyfluoran-β-D-galactoside (10) 6-bromo-2-naphthyl-β-D-galactoside (11) 2-7-thiyl-β-〇-galactoside (1
2) Fluorescein-di-β-logalactoside In addition, signal detection can be facilitated by further reacting the above-mentioned liberated aglycone with another substance. For example, 5-bromo-4-chloro-3-indolyl-β-D
- When using galactoside, a preferred method is to react the resulting liberated aglycone with a tetrazolium salt as follows.

(1) 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H tetrazolium chloride (2) 3-(4,5-dimethyl-2-thiazolyl)
-2,5-diphenyl-2H tetrazolium peptide (
3) 3.3'-(4,4'-biphenylene)-bis(2,5-diphenyl-2H tetrazolium chloride) (4) 3.3'-(3,3'-dimethoxy-4,4
'-biphenylene)-bis(2-(p-nitrophenyl)-5-phenyl-2H tetrazolium chloride) (5) 3.3'-(3,3'-dimethoxy-4,4
'-biphenylylene)-bis(2,5-diphenyl-2
H tetrazolium chloride) (6') 3.3'-(3,3'-dimethoxy-4,
4'-biphenylylene)-bisC2,5-bis(p-nitrophenyl)-2H tetrazolium chloride] Alternatively, aryl or alkyl-β-D-
Using galactoside as a substrate, the liberated galactose is conjugated with galactose oxidase (EC1, 1, 3, 9) and the generated hydrogen peroxide is measured by various known methods, or the liberated galactose is conjugated with hexokinase (EC2, 7). , 1, 1) to measure the decrease in TP or increase in ADP, or the free galactose can be conjugated to galactose dehydrogenase (EC1, 1, 1).
, 48 or 1.1.1.120) to form NAD+
or decrease in NADP+, or NADH or NADPH
Measuring the increase in is also a preferred method.

In this specification, the substrates, tetrazolium salts, enzymes used in the conjugation reaction, coenzymes, etc. required for measuring the enzymatic activity of β-D-galactosidase are collectively referred to as an activity measuring reagent.

The specific application method of the activity measuring reagent varies depending on each aspect of the present invention, but may follow the description of each of the above-mentioned disclosed techniques corresponding to each aspect.

Substances that can be used in the present invention and which are capable of binding to specific components or analogs thereof in fluid samples include antibodies, antigens, lectins, protein A, specific enzyme inhibitors, etc., depending on the target to be measured. It is particularly preferable that the binding reaction between the specific component and the binding substance is an antigen-antibody reaction. The origin of the antibodies used in the present invention is not particularly limited, and antiserum obtained by administering and immunizing mammals with antigens, ascites fluid as it is, or sodium sulfate using a conventionally known method. It can be purified and used by a precipitation method, an ammonium sulfate precipitation method, a gel filtration method using Sephadex gel, an ion exchange cellulose chromatography method, or an electrophoresis method (see "Immunochemistry" edited by Shunsuke Migita, Kabun Shoten; pp. 74-88).

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

In addition, these antibodies are IgG, IgM, IgA, I
gD and IgE can be used individually, or these antibodies can be treated with enzymes to produce Fab, Fab' or F(a
b'), etc. may be used in the form of active antibody fragments. 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 present invention belongs to the immunoassay method, and its reaction types include the competitive method, the two-antibody method, and the Sand-Deutsch method. It will be done. Since the measurement method of the present invention can be particularly preferably used in immunoassay methods, the present invention will be described in detail below using an immunoassay method as an example. However, the present invention is not limited to this explanation, and can be applied in various ways. It is clear from what has been described above that this is possible.

The carrier-immobilized substance used in the present invention includes the Sand-Deutsch method, and has component a of the analysis system, a substance that specifically binds to a specific component, a substance that can specifically bind to a substance bound to the carrier, and There are two types: a carrier-immobilized product in which a substance capable of specifically binding to the specific component is selected, and a carrier-immobilized product of an arylmercury derivative immobilized on the carrier.

The reason why two immobilized substances are used in this way is that when a specific component or a substance that can specifically bind to the specific component and an enzyme or an arylmercury derivative form a binding species with one immobilized substance, the other immobilized substance forms a binding species with the other immobilized substance. This is to prevent binding to the immobilized material. However, in this case, if one is used without being fixed, it will further bind to the bound species, making measurement impossible. The immobilized product of the present invention can be prepared by physically adsorbing these substances to the surface of a carrier used in affinity chromatography, immobilized enzymes, or immunoassays by various known methods, or directly or indirectly by chemical reaction. It is created by combining the At that time, it is necessary to take care not to lose the specific binding property of the substance to the specific component. , published in 1978) and some parts of Chibata,
The method described in "Experiments and Applications Affinity Chromatography" by Tetsuya Tosa and Yuji Matsuo (published by Kodansha, 1976) can be cited as an example of a preferred method.

Furthermore, in the case of aryl mercury derivatives, they do not need to be in a free or dissolved state in an aqueous medium; they may be dispersed in an insoluble state in an aqueous medium, and they can also be used in oil protection dispersions or lipids used in the production of color photographs. It may be contained in a bimolecular chain. In addition to the above-mentioned methods, methods used for producing immobilized enzymes, such as "Immobilized Enzymes" edited by Chibata (published by Kodansha, 1981), may also be mentioned.

Examples of carriers used in the present invention include dextran polymer, agarose, cellulose, gelatin, acrylamide, glass beads, polystyrene beads, and
Granular structure for transportation described in No. 90859, JP-A-57-1
No. 01760, No. 57-101761, No. 58/70
Examples include self-bonding particle combinations and fibrous porous materials, which are described in 163 Myo et al.

When the immobilized product 1; -7 forms a binding species, it is sufficient that the immobilized product is immobilized so as not to bind to the other immobilized product, and the carrier immobilization method is not particularly limited.

Further, more detailed explanations of these carriers and immobilized substances are described in each of the above-mentioned disclosure documents.

The preferred thickness of each layer constituting the analytical element of the present invention is not determined solely by its function, but varies depending on the material constituting the layer and the presence of other layers.

When the constituent material is mainly granular, the porous reaction layer essential to the present invention has a diameter of 3 to 700 μm, and further 10 to 30 μm.
A film thickness of 0 μm is desirable. Furthermore, according to an aspect of the invention, if there are multiple porous reaction layers mainly composed of granules, or if there is a porous layer composed of granules in other functional layers such as a spreading layer, all The total thickness of the porous layer consisting of the granules is in the range of 10 to 7002 m1, more preferably 20 to 300 m.

When the porous reaction layer is mainly composed of fibers, it is preferably 20-1200 μm1, more preferably 40-70 μm1.
It is in the range of 0μ turtle. Similarly, when a porous layer made of fibers is present in a functional layer such as another spreading layer, the total thickness of all the porous layers made of fibers is in the range of 40 to 1200 μm, more preferably 100 to 400 μm. .

Furthermore, when both the granular porous layer and the fibrous porous layer are present, the total porous layer is in the range of 60 to 1500 μl, more preferably 100 to 900 μl.

In addition, coloring reagent layers, binding anastomosis layers, label-containing layers, etc.
When layers consisting of a continuous binder are present, the thickness of these layers is 5 to 200 μm, more preferably lO to 100 μm.
- is in the range. Furthermore, the total thickness of the layer consisting of the continuous binder is 5 to 200 μm, and more preferably 10 to 100 μm.
It is desirable that it be within the range of .

As an exception, it is preferable that the adhesive layer, protective layer, and timing layer be as thin as possible as long as the desired effect can be achieved, and there is no lower limit to the thickness.

〔Example〕

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

Example 1 Preparation of l-(1) p-aminophenylmercuric acetate (enzyme inhibitor) immobilized Avicel
0) Add to 300Q, suspend and add pure water (
deionized wood) cyanogen bromide 80 dissolved in 800a+Q
．． After 20 minutes of reaction, the mixture was washed thoroughly with water. 80 g of this Avicel was suspended in 95QmQ of a 25% dimethyl sulfoxide aqueous solution containing 4.8 g of p-aminophenyl mercuric acetate, and stirred at room temperature for 20 hours. This was collected by filtration, washed with dimethyl sulfoxide and pure water, and then washed with 1M Tris-HCl buffer (pH 8-5) 1
000a12 and stirred at room temperature for 20 hours to block unreacted groups. After filtering this and washing thoroughly with water,
Collected by filtration, thoroughly washed with water, washed with acetone and dried to obtain p-
Avicel fixed with aminophenyl mercuric acetate was prepared.

1-(2) Preparation of avidin-immobilized Avicel Bovine serum albumin (Fraction V; manufactured by Arma, USA) 1.
Og was dissolved in 0.01M phosphate buffer (pH 7.4) 330aQ containing 0.15M sodium chloride, and dimethylformamide containing 4og of biotin-N-hydroxysuccinimide ester (manufactured by Boehringa) IO was added to the solution. After reacting for 2.5 hours at room temperature in addition to 3.0 ml (l) of the solution,
Biotinylated bovine serum albumin was obtained by thorough dialysis against the above buffer solution and freeze-drying.

Next, 90 g of Avicel (manufactured by Asahi Kasei Corporation) was suspended in 1800 mQ of 2.5 M phosphate buffer (PT112.0), and 45.0 g of cyanogen bromide dissolved in 550 md of pure water was added to this under ice water cooling. After reacting for 20 minutes, it was collected by filtration and thoroughly washed with water.

90 g of this Avicel was mixed with 0.1 M hydrogen carbonate and thorium aqueous solution (containing 0.15 M sodium chloride) containing 500 mg of the above biotinylated bovine serum albumin.
2 and stirred at 4°C for 20 hours. This was collected by filtration and washed alternately with pure water, a 0.1M aqueous sodium bicarbonate solution containing 0.15M sodium chloride, and a 0.1M sodium acetate buffer (pH 4,1) containing 0.15M sodium chloride. , 1M Tris-HCl buffer (pH 8,5
) 1200I and stirred at room temperature for 20 hours to block unreacted groups. After filtering this and washing thoroughly with water,
Avidin (manufactured by Oriental Yeast Co., Ltd.) 270mg was dissolved in O, 15M sodium chloride-containing 0.05M phosphate buffer (pt(7,4) 45011 (2), suspended in 4°
After reacting at C for 200 hours, the mixture was collected by filtration and washed with water to produce Avicel with avidin immobilized thereon. Furthermore, 240I of pure water containing 15.4g of bovine serum albumin and 9.91g of sucrose was added.
80 g of the above avidin-immobilized Avicel was suspended and lyophilized to prepare avidin-immobilized Avicel containing bovine serum albumin and sucrose.

1-(3) Preparation of β-galactosidase-labeled human IgG Human IgG (manufactured by Kappel, USA) 20 mg to 0.1
M phosphate buffer (PH6,5) 2.011112 was dissolved in the solution, and 2.5 mg/ml of N-(ε-maleimidocagloyl roquine) succinimide (manufactured by Isotope Kagaku Kenkyusho) was added thereto.
2 Add 77 tt (l) of dimethylformamide solution to 30
°C! - After reaction for 20 minutes, SmMEDTA containing 0.1
Maleimidized human Ig purified with a Sephadex G-25 column equilibrated with M phosphate buffer (pH 6,0)
I got a G. Next, β-galactosidase (manufactured by Toyobo)
lo, 5mg/mI 20.1M phosphate buffer 1.8t
all 2, the maleimidized human IgG 13.6
After reacting at 4°C for 45 hours with the addition of 3.2 raQ of a solution containing 0.1 M 2-megabutethylamine! 7
Add 5 μff and react for 20 minutes at 30°C, and add 0.1M phosphate buffer containing 0.15M sodium chloride (pFI7.4).
β-galactosidase-labeled human IgG was obtained by separation and purification using a Superose 6 prep grade (manufactured by Pharmacia) column equilibrated with t;

1-(4) Preparation of biotinylated goat anti-human IgG antibody Anti-human KgG antibody (manufactured by Kappel, USA) 5.8m
g was dissolved in 0.1M phosphate buffer (pH 7,4) containing 0.15M sodium chloride (2.0+m4+), and this contained 0.32mg of biotin-N-hydroxysuccinimide ester (manufactured by Boehringa). Add 1500 μa of dimethylformamide m and react at room temperature for 3.0 hours, then thoroughly dialyze against 0.01 M phosphate buffer containing 0.15 M sodium chloride to remove biotinylated goat anti-human Ig.
G antibody was obtained.

1-(5) Creation of immunological analysis element Thickness: 180 μm
A coating liquid (1) having the following composition was applied and dried on a transparent undercoated polyethylene terephthalate film to form a gelatin layer.

Coating liquid - (1) Deionized gelatin 6.0g Triton X-100 (manufactured by Rohm and Haas) O, 15g1
．． 0.01 g of 2-bis(vinylsulfonyl)ethane 54.0 g of pure water Next, the p-aminophenylmercuric acetate-immobilized Avicel prepared in 1'-(1) above was dispersed, and the following solution containing a coloring reagent was added. A coating solution of composition (2) is applied onto the gelatin layer and dried.

Coating liquid - (2) p-aminophenyl mercuric acetate immobilized Avicel 14.0g Triton
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Behringa) 0.90g
NeoTB (manufactured by Isotope Chemical Research Institute) 0.3
5gn-butanol 34.0
g* NeoTB: 3.3'-(4,4'-biphenylene)-bis (2,5-diphenyl-2H tetrazolium chloride) Next, the avidin-immobilized Avicel prepared in 1-(2) was dispersed, and the following A coating solution of composition (3) was applied onto the p-aminophenylmercuric acetate-immobilized Avicel layer and dried.

Coating liquid - (3) Bovine serum albumin/sucrose-containing avidin-immobilized Avicel 22.5g Triton X-1002.5g Polyvinylpyrrolidone 3.50gn~
52.0 g of butanol Furthermore, a coating liquid (4) having the following composition was coated and dried on the avidin-immobilized Avicel layer to form a spreading layer.

Coating liquid = (4) Powder filter paper D (manufactured by Toyo Roshi Co., Ltd.) 30.0 g Triton As an analytical element.

1-(6) Measurement of human IgG A human IgG solution (0 to 64
0 μg/IIIQ), β-galactosidase-labeled human IgG prepared in 1-(3) (10 μg/1a (above buffer solution in 1)) and biotinylated anti-human+gc antibody prepared in 1-(4) (20/J After mixing with g/Io (l of the above buffer solution), this mixed solution f'4LOμQ was mixed with 1-(5
), and after incubation in a sealed state at 37°C for IO minutes, 546 nm was applied from the support side.
The reflection density of m was measured. The results are shown in Figure 1.

Example 2 2-(1) Creation of avidin-immobilized cellulose fibers In the same manner as above, using powder filter paper D (manufactured by Toyo Roshi Co., Ltd.) in place of the avidin-immobilized Avicel prepared in 1-(2) above. Avidin-immobilized cellulose fibers containing bovine serum albumin and sucrose were prepared.

2-(2) Creation of immunological analysis element Thickness: 180 μm
A coating liquid (5) having the following composition was applied and dried on a transparent undercoated polyethylene terephthalate film to form a gelatin layer.

Coating liquid - (5) Deionized gelatin 4.5g) Rydon X-1000, 10g 1.2-bis(vinylsulfonyl) ethane 0.007gN
eoTB
0-23g pure water
40.5g Next, the coating liquid described in 1-(5)-(2)
A dispersion having the composition from 1 to 1, except for NeoTB, was applied onto the gelatin layer and dried to form a p-aminophenylmercuric acetate-immobilized Avicel layer.

Further, on this layer, a coating liquid (6) having the following composition was applied and dried, and then cut into a size of 1.5 x 1.5 cm'' to obtain an analysis screen.

Coating liquid - (6) Bovine serum albumin/sucrose-containing avidin-immobilized powder filter paper D 30.0g Triton
3) Measurement of human IgG Human IgG was measured in the same manner as in 1-(6). The results are shown in FIG.

Example 3 3-(1) Creation of immunological analysis element Thickness: 180 μm
The coating solution (5) described in 2-(2) was applied and dried on the transparent undercoated polyethylene terephthalate film to form a gelatin layer. Next 1-(5)
A dispersion obtained by removing NeoTB from the coating liquid (2) described above was applied onto the gelatin layer and dried to form a p-aminophenylmercuric acetate-immobilized Avicel layer.

Furthermore, on top of this layer, a coating solution with the following composition containing the β-galactosidase-labeled human IgG prepared in 1-(3) and the biotinylated goat anti-human IgG antibody prepared in 1-(4) After applying and drying 7), and further applying and drying the coating liquid described in 2-(2)-(6) on this layer, it was cut into a size of 1.5 x 1.5 cm, and an analytical element was prepared. And so.

Coating solution - (7) Orchid serum albumin/sucrose-containing avidin-immobilized Avicel 22.5g Triton X-1002, 50g Polyvinylpyrrolidone 3.30gβ-
Galactosidase-labeled human Ig human O, 16mg biotinylated goat anti-human IgG antibody o, 6omg serum albumin 38.0mg n-butanol 55.0g3-(2)
Measurement of human IgG Various 0.3M buffers shown in the attached table containing 11M magnesium chloride and 6vt% bovine serum albumin (
Human IgG solution dissolved in pi(7,2) (0-640
pg/ra12) was dropped onto this analytical element, and 37°01
After incubation in a sealed state for 0 minutes, the reflection density at 546 nm was measured from the support side. The results are shown in Figure 3.
It was shown to.

Example 4 4-(1) β-galactonase-labeled rabbit anti-human virus)
Preparation of 1gG antibody Rabbit anti-human IgG antibody (manufactured by Kappel, USA) 20mg
A β-galactosidase-labeled rabbit anti-human IgG antibody was prepared using the same method as in 1-(3).

4-(2) Measurement of human IgG Human IgG solution (0-640p g/m12) dissolved in various 0 and 3M buffers (pH 7,2) containing laM magnesium chloride and 6% bovine serum albumin.
The above buffer solution of β-galactosidase-labeled rabbit anti-human IgG antibody (15 μg/n+4) prepared in 4-(1)) and the biotinylated goat anti-human IgG antibody prepared in 1-(4) (100 μg) /rnQ buffer solution), 10 μa of this mixed solution was dropped onto the analytical element prepared in 1-(5), and after incubation in a sealed state for 37°O1O minutes, the reflection density at 546 nm was measured from the support side. It was measured. The results are shown in FIG.

Example 5 5-(1) Creation of Avicel immobilized with goat anti-human IgG antibody 90 g of Avicel (manufactured by Asahi Kasei Corporation) was dissolved in 2.5 M phosphate buffer (
Add cyanogen bromide dissolved in 550 ml (l) of pure water to this suspension under ice-water cooling.
．． After 20 minutes of reaction, the mixture was collected by filtration and thoroughly washed with water. 900 m of a 0.1 M sodium bicarbonate aqueous solution (containing 0.15 M sodium chloride) containing 90 g of this Avicel and 600 mg of goat anti-human IgG antibody (manufactured by Kappel)
ff and stirred at 4°C for 20 hours. This was collected by filtration and washed alternately with pure water, a 0.1M aqueous sodium bicarbonate solution containing 0.15M sodium chloride, and a 0.1M sodium acetate buffer (pH 4,1) containing 0.15M sodium chloride. , IM) Lis-HCl buffer (pH 8,5
) 1200 m (2) and stirred at room temperature for 20 hours to block unreacted groups. After filtering this and thoroughly washing with water, 15.4 g of orchid serum albumin and 9.0 g of sucrose were added.
80 g of the solid antibody Avicel was suspended in 240 I pure water in which 91 g of the antibody was dissolved, and lyophilized to prepare Avicel immobilized with a goat anti-human IgG antibody containing bovine serum albumin and sucrose.

5-(2) Preparation of immunological analysis element In 1-(5), 22.5 g of bovine serum albumin/sucrose-containing avidin-immobilized Avicel of the coating solution (3) was added to the bovine serum albumin prepared in 5-(1). Goat anti-human Ig containing serum albumin and sucrose
A completely similar analytical element was prepared except that 22.5 g of G antibody-immobilized Avicel was used instead.

5-(3) Measurement of human IgG Various 0.3M buffers (p) 1 shown in the attached table containing 1mM magnesium chloride and 6% bovine serum albumin
7.2) Human IgG solution (0-640μg
/mQ), is mixed with β-galactosidase-labeled human IgG prepared in 1-(3) (10 μg/iff of the above buffer solution), and 10 μa of this mixed solution is dropped onto the analytical element prepared in 5-(2). After incubation at 37° C. for 10 minutes in a sealed state, the reflection density at 546 nm was measured from the support side. The results are shown in FIG.

Example 6 6-(1) Measurement of human IgG Various 0.3M buffers (pH 7) shown in the attached table containing 1mM magnesium chloride and 6% bovine serum albumin
, 2) human IgG solution (0 to 640 μg/
mQ) with the β-galactosidase-labeled rabbit anti-human IgG antibody prepared in 4-(1) (15 μg/vaQ of the above buffer solution), 10 μa of this mixed solution was
-Created in (2). Drop onto the analytical element and heat to 37°C1O
After incubation in a sealed state for a minute, the reflection density of 546 na was measured from the support side. The results are shown in FIG.

Table 〉 Various concentrated liquid compounds used for performance evaluation A1: Bis (2-7 droxyethyl) iminotris (hydroxymethyl) methane ([(OCI(,), CN(CH2Cl, OH).

Compound B1 Nidris (hydroxymethyl)aminomethane (HOCHz)scNHz Compound CI = l-sodium phosphate-2-sodium phosphate Compound D+: N-tris (hydroxymethyl)methyl-
2-Aminoethanesulfonic acid (HOCHt)scNHcHxcHzsOsH Compound E
, :3-(N-1-lis(hydroxymethyl)methylaminoco-2-hydroxypropanesulfonic acid (HOC)12), CNHCI(, CHCH2So, H
■ OH Compound Fr: 3-CN,N-bis(2-hydroxyethyl)aminoco-2-hydroxypropanesulfonic acid ()10CH,C)! ,)2NC)I2CHCH2SO
Only the UHOH compound A1 is used in the method of the invention.

Compound B l+c l is a comparative example of a commonly used compound.

Compounds D +, E r, F + are comparative examples of similar compounds.

Compounds A, D, E, and F were all manufactured by Isotope Kagaku Kenkyusho.

Looking at the above results, it can be seen that the buffer Al based on the method of the present invention
It can be seen that when B+ was used, the results were much better than when the commonly used buffers B+ and C1 were used.

Moreover, compared to similar compounds Dr, E +, F r, A
Case 1 is considerably better, and it is clear that the effects of the present invention can be obtained only from so-called good buffers having a specific structure.

Example 7 7-(1) Preparation of Avicel immobilized with p-aminophenylmercuric acetate (enzyme inhibition column) The same method as in 1-(1) in Example 1 above was used.

7-(2) Preparation of avidin-immobilized Avicel 1-
It was created in exactly the same manner as (2).

7-(3) Preparation of β-galactosidase-labeled human IgG It was prepared in exactly the same manner as in 1-(3) above.

? -(4) Preparation of biotinylated goat anti-human IgG antibody It was prepared in exactly the same manner as in 1-(4).

7-(5) Creation of immunological analysis element Exactly the same method and coating solution as in 1-(5) above-(1),
Using (2), (3') and (4), 1.5X 1.5
An analytical element with a size of 1 cm" was obtained.

7-(6) Measurement of human IgG 1-(
The performance of the analytical element was checked in exactly the same manner as described in 6), and the results are shown in FIG.

Example 8 8-(1) Preparation of avidin-immobilized cellulose fiber It was prepared in exactly the same manner as in 2-(1) above.

8-(2) Preparation of immunological analytical element 2-(2) above
Obtain an analytical element in exactly the same manner as above.

8-(3) Measurement of human IgG Human 1gG was measured in the same manner as in 1-(6). The results are shown in FIG.

Example 9 9-(1) Preparation of immunological analytical element An analytical element having a size of 1.5×1.5CO+' was obtained in exactly the same manner as in 3-(1).

9-(2) Measurement of human IgG The performance of the analytical element was checked under exactly the same conditions as in 3-(2), and the results are shown in FIG.

Example 10 10-(1) Preparation of β-galactosidase-labeled rabbit anti-human gG antibody It was prepared under the same conditions as in 4-(1).

1O-(2) Measurement of human IgG Measurement was performed under the same conditions as 4-(2), and the results are shown in Figure 1O.

Example 11 11-(1) Preparation of goat anti-human IgG antibody-immobilized Avicel was prepared in the same manner as in 5-(1).

1l-(2) Preparation of immunological analytical element An analytical element completely similar to 5-(2) was prepared.

1l-(3) Measurement of human IgG Reflection density at 546 nm was measured under the same conditions as in 5-(3), and the results are shown in FIG.

Example 12 12- (1) Measurement of human IKG The reflection density at 546 nm was measured from the support side in exactly the same manner as in 6-(1), and the results are shown in FIG.

Classification table The compounds used for evaluation of the present invention are as follows.

Compound A2: Piperazine-N,N'-(2-ethanesulfonic acid) Compound B! :N-2-hydroxyethylpiperazine-N
'-2-ethanesulfonic acid compound C, nidris (hydroxymethyl)aminomethane ()IOCH,)3-C-doH Compound: l-sodium phosphate-disodium phosphate compound E! :3-CN-1-lis (hydroxymethyl)methylaminoco-2-hydroxypropanesulfonic acid (I(OCI(z)sCNHCHzCHCHzSOsH
H Compound F, +3-(N,N-bis(2-hydroxyethyl)amine]-2-hydroxypropanesulfonic acid ()10c11ICHI), NC)1. c) ICHIS
O,HH Compounds A, B are examples of the present invention Compounds C2 and D2 are comparative examples of commonly used compounds Compounds E,, F are comparative examples of similar compounds Compounds A, B, E, F , used those manufactured by Isotope Kagaku Kenkyusho.

Looking at the above results, it can be seen that buffer A based on the method of the present invention
,,B, when using the commonly used buffer C,
It can be seen that the results are much better than when using ,D.

Moreover, similar compound E! , F , the case of A2 is considerably superior, and it is clear that the effects of the present invention can be obtained only from the so-called good buffer solutions having a specific structure.

Example 13 13-(1) Creation of epoxy group-introduced Avicel 100 g of Avicel was mixed with butanediol diglycidyl ether (
Aldrich) 250mQ and 2 vag/II
Suspended in a mixture with 0.6M sodium hydroxide aqueous solution 250111ff containing sodium borohydride of IQ,
After shaking and stirring at 40° C. for 5.5 hours, the mixture was washed with 5Q pure water and dried.

13-(2) Preparation of phenyl-β-D-thiogalactopyranoside-immobilized Avicel 1.5 g of phenyl-β-〇-thiogalactopyranoside was dissolved in Q, IM Na0tl 6(b(1). ,
Add 20g of Avicel prepared in 13-(1) and heat at 45°C.
The mixture was shaken and stirred for 16 hours.

After completion of the reaction, filter, water, 2.5% glucose solution, water,
0.1M acetate buffer (pH 4) containing 0.5M NaC (l)
, 0), and then washed with water and dried.

13-(3) Preparation of 5-aminoisophthalic acid immobilized Avicel 20g of 5-aminoisophthalic acid was dissolved in 2N NaOH1
30 g of Avicel prepared in 13-(1) was dissolved in 1Oa+Q, and the mixture was shaken and stirred at 40°C for 20 hours.

After completion of the reaction, filter and add 0.2N NaOH, water, 0.1M
It was washed successively with acetate buffer (pH 4.0), water, 0.1M sodium bicarbonate containing 0.5M NaCQ, and water, and then dried.

+3- (3') Preparation of Avidin-immobilized Avicel and goat anti-human IgG antibody-immobilized Avicel 90 g of Avicel (manufactured by Asahi Kasei) was dissolved in 2.5 M phosphate buffer (
pH 12,0) 1800 m (Additionally to 2, suspend and cool with ice water, add cyanogen bromide 4 dissolved in 550 mQ of distilled water.
After adding 5.0 g and reacting for 20 minutes, it was collected by filtration and thoroughly washed with water. 90g of this Avicel was mixed with 900ml of an O, 1M aqueous sodium bicarbonate solution (containing 0.15M sodium chloride) containing 270mg of avidin (manufactured by Oriental Yeast Co., Ltd.) or 50mg of goat anti-human IgG antibody (manufactured by Kappel Co., Ltd.).
Q and stirred at 4°0 for 20 hours. This was collected by filtration and washed alternately with distilled water, a 0.1M aqueous sodium bicarbonate solution containing 0.15M sodium chloride, and a 1M sodium acetate buffer (pH 4,1) containing 0.15M sodium chloride. , LM) Lis-HCl buffer (pH 8,
5) It was suspended in 12001117+ and stirred at room temperature for 20 hours to block unreacted groups. This was collected by filtration, thoroughly washed with water, and then filtered, washed with water, and lyophilized to produce avidin-immobilized Avicel and goat anti-human IgG antibody-immobilized Avicel.

13-(4) Preparation of glutamate dehydrogenase-labeled rabbit anti-human IgG Glutamate dehydrogenase (EC1, 4, 1, 4
ProLeus sp, origin) 1.25% lomg
0.1M phosphate buffer (pH 6) containing glutaraldehyde
, 8) 0.2+n4j: was added and allowed to react by gently stirring at room temperature for 10 hours. This was passed through a Sephadex G-25 column (1.0 x 55 cm) equilibrated with 0.15M sodium chloride solution in advance, and a glutaraldehyde-activated glutamate dehydrogenase fraction was collected. To 1.0 Il (l) of this fraction, add 1.0 mu of O, 15 M sodium chloride solution containing 5 II 1 g of rabbit anti-human IgG (manufactured by Kappel, USA), and 0.1 mff of 1 M sodium carbonate buffer (pH 9,5). The reaction was carried out at ℃ for 24 hours.To this solution was added 0.1 mQ of 0.2M lysine.
After further reaction at 4°C for 2 hours, O, 15M
0.01M phosphate buffer containing sodium chloride (pH 7,
Lllt-rogel AcA-3- equilibrated with 6)
4 columns (1,5X 100c+a) to obtain glutamate dehydrogenase-labeled rabbit anti-human IgG.

13-(5) Creation of analysis element A dispersion liquid having the following composition was prepared.

Coating liquid (1) p-aminophenyl mercuric acetate immobilized Avicel 14.0 g Triton
Butanol 34.0g Coating solution (2) Avidin-immobilized Avicel 14.0g Triton
-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Behringa) 0.90g
NeoTB (manufactured by Isotope Chemical Research Institute) 0.3
5gn-butanol 34.
0g* NeoTB: 3.3'-(4,4'-biphenylene)-bis (2,5-diphenyl-2H tetrazolium chloride) Coating solution (3) Avidin-immobilized Avicel 14.0g Triton X -too 1.4g Polyvinyl Pyrrolidone (manufactured by Wako Tsumugi Pharmaceutical) 1.2g Chlorophenol red-β-D-galactopyranoside (CPRG) (manufactured by Boehringer Mannheim) 0.90g
n-butanol 34.0g
Coating liquid (4) Avidin-immobilized Avicel 14.0g) 5
1.2go-nitrophenyl-
β-D-galactopyranoside (Merck) 0.90gn
-Butanol 34.0g Coating solution (5) Avidin-immobilized Avicel 14.0g Glutamic acid 030g 1-Methoxy-5-methylzenadinium methyl sulfate 10mg Oxidized nicotinamide adenine dinucleotide phosphate 4omg NeoT
B (manufactured by Isotope Kagaku Kenkyusho) 0.35g Triton
Butanol 34.0g coating solution (6) Powder filter paper D (manufactured by Toyo Roshi Co., Ltd.) 30.0g Triton .0g Triton
Bromo-4-chloro-3-indolyl-β-D-galactopyranondo (Behringa) 0.90g
NeoTB (manufactured by Isotope Research Institute) 0,
35gn-butanol 34
．． 0g wood NeoTB: 3.3'-(4,4'-biphenylene)-bis (2,5-diphenyl-2H tetrazolium chloride) The above coating solution was applied onto a transparent subbed polyethylene terephthalate film with a thickness of 180 μm. The samples were coated and dried in the following order to prepare analytical elements A to D.

Each element was cut into a size of 1.5×1.5c+a'' and used as an analytical element.

13-(6) Human IgG measurement A reagent solution with the following concentration composition was prepared.

Reagent solution a 1% Bovine serum albumin 5% Collagen vegtide A (cow bone) (manufactured by Funakoshi Pharmaceutical) 0.3M Bis(2-hydroquinethyl)iminotris (hydroxymethyl) Methane buffer (pH 7,2) 1mM Magnesium chloride ■5μg/IaQp5μLactosidase drifting rabbit anti-human IgG antibody 100μg/mQ Biotinylated goat anti-human IgG antibody reagent solution b 1% Bovine serum albumin 5% Collagen vegtide A (cow bone) (manufactured by 7 Nakosi Pharmaceutical) 0.3M Sodium phosphate buffer (pH 7,2) 1m
lj Magnesium chloride 15 μg/ml 12p-galactosidase labeled rabbit anti-human IgG antibody 100267m (l Biotinylated goat anti-human IgG antibody reagent solution C 1% Bovine serum albumin 5% Collagen vegtide A (cow bone) (manufactured by Funakoshi Pharmaceutical) (], 33M N-2-hydroxyethylpiperazine=
N'-2-Hydroquine globan-3-sulfonic acid buffer (pH 8,5) lmM Magnesium chloride/Rum I5pg/mQ Glutamate dehydrogenase labeled rabbit anti-human IgG antibody 100μg/m4 Biotinylated goat anti-human IgG antibody reagent solution d 1% bovine serum albumin 5% collagen peptide A (cow bone) (manufactured by 7 Nakosi Pharmaceutical) 0.3M potassium phosphate buffer (pH 8.5) 1, m
M Magne chloride/Rum 15μg/IIIQ, Rabbit anti-human IgG antibody for glutamate dehydrogenase detection 100μg/mQ Biotinylated goat anti-human IgG antibody reagent solution e 1% False serum albumin 5% Collagen peptide A (cow bone) (manufactured by Funakoshi Pharmaceutical) 0.3M bis(2-hydroxyethyl)iminotris (hydroxymethyl) methane buffer (pH 7,2) ImM Magnesium chloride 15μg/mQ p-galactonase Keyaki rabbit anti-human IgG antibody Also IgG-depleted human serum (manufactured by Miles) Alternatively, 640 μg/ll1a of human IgG dissolved therein was prepared as a sample.

Next, according to the combination shown in the table below, mix 4 parts of the reagent solution with 1 part of the sample and immediately drop it onto the analytical element for 20 minutes.
After incubation at 7°C, reflection density was measured.

Table Dr@. : Reflection density when measuring IgG 640 μg/mα sample Dro : IgG OI1
Reflection density when measuring a sample of g/m12 Further, Examples and Comparative Examples are classified and grouped as follows: (1) Labeling substances that use the inhibitor or buffer according to the present invention and have particularly high effects of the present invention There are Nos., 1', 2 and 9 as cases where a chromogenic substrate is used, and Comparative Example No.

3 and 4 are the corresponding groups; (2) No. 5 is a case in which the inhibitor and buffer according to the present invention are used, and a particularly highly effective labeling substance of the present invention is used; a group corresponding to the comparative example; , (3) N007, which satisfies at least the requirements of the present invention, is roughly divided into the group of Comparative Example 8, and when compared within each group, the effects of the present invention are obvious.

〔Effect of the invention〕

Looking at the above results, when the buffer solution based on the method of the present invention is used, it is possible to give much better results than when the normally used buffer solution is used.

Furthermore, the buffer according to the present invention is considerably superior to similar compounds, and a particularly suitable buffer among the so-called good buffers has been identified.

[Brief explanation of the drawing]

1 to 12 are graphs showing measurement results using an analytical element embodying the analytical method of the present invention. Junjin Konica Co., Ltd. Compound AI (compound of the present invention) ---1---・------ Compound Bl (compound of the present invention) ----・〜・・・・・・−
・・Compound Ct (comparative example)-1---------・--
1-・～・ Compound Dl! (Comparative example) □ Compound AI (
Compound of the present invention) ---Compound Bl (Compound of the present invention)--1 Compound E , (Comparative example)-----11----
--- Compound F, (comparative example) □ Compound A, (compound of the present invention) -111 --- Compound B2 (compound of the present invention) --m −−−−−−・−・−−−−−
・ Compound C, (comparative example) -1
--- Compound B2 (compound of the present invention) Compound At (compound of the present invention) --- Compound B2 (compound of the present invention) --- Compound B2 (compound of the present invention) -m--・--・-- Compound Ea (comparative example)-11111・---------------------
Compound F2 (comparative example) □ Compound AI (compound of the present invention) -----...--1--Compound Bl (compound of the present invention)...-111--... −・・・−・・−・
Compound ci (comparative example)---111------
--- Compound (comparative example) □ Compound A+ (compound of the present invention) ----...--1 --- Compound Bl (compound of the present invention) --- −−One・・・・・・−・・−
・Compound E, (comparative example) =----------------------
--- Compound p+ (comparative example) Compound Ai (compound of the present invention) --- Compound Bl (compound of the present invention) ---・−・・−・・−・−・
- Compound C, (comparative example) -----11 -------
--- Compound D2 (comparative example) □ Compound AI (compound of the present invention) -1.--.--1 Compound B, (compound of the present invention) -
1. Compound at (comparative example) 1.
Compound Fl (comparative example) −・・−・−・−・・−・・−
Compound B2 (compound of the present invention)-----
--111・ Compound Fg (comparative example) □ Compound Al (
Compound of the present invention) -・-・・-・・−・・−・・Compound Bl (Compound of the present invention)
・Compound C1 (comparative example) -------
--- Compound 0. (Comparison 91) □ Compound A, (compound of the present invention)

Claims (16)

[Claims] (1) In the analysis process of a specific component in a fluid sample, a, a substance that specifically binds to the specific component, which is bound to either a biologically active substance that does not bind to the specific component, or a substance that specifically binds to the biologically active substance; b. Either a labeled body formed by binding a labeling substance to the specific component or its analog, or a labeled body formed by binding a labeling substance to a substance that specifically binds to the specific component, and c. At least one type. The carrier contains either a substance that specifically binds to the biologically active substance or a biologically active substance that does not bind to the specific component, and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance. A method for analyzing the specific component using an analysis system formed from a combination of porous reaction layers contained separately immobilized on separate carriers, wherein the specific component is specifically bound to the labeling substance and modulates the signal caused by the labeling substance. 1. A method for immunologically analyzing a specific component in a fluid sample, wherein the substance is an arylmercury derivative and further contains a compound represented by the following general formula [I] and/or a salt thereof. General formula [I] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R_1, R_2, R_3, R_4 and R_5 represent an alkyl group with 1 to 5 carbon atoms having a hydroxyl group or sulfone group as a substituent in the main chain. . ] (2) The compound represented by the general formula [I] is bis(
2. The method for immunologically analyzing a specific component in a fluid sample according to claim 1, wherein the component is 2-hydroxyethyl)iminotris(hydroxymethyl)methane. (3) In the analysis process of a specific component in a fluid sample, a, a substance that specifically binds to a specific component, which is bound to either a biologically active substance that does not bind to the specific component, or a substance that specifically binds to the biologically active substance; b. Either a labeled body formed by binding a labeling substance to the specific component or its analog, or a labeled body formed by binding a labeling substance to a substance that specifically binds to the specific component, and c. At least one type. Either a substance that specifically binds to the biologically active substance or a biologically active substance that does not bind to the specific component, and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance are added to the carrier. In a method of analyzing the specific component using an analysis system formed from a combination of porous reaction layers contained and immobilized separately on a carrier in the form of a carrier, the method specifically binds to the labeling substance and modulates the signal caused by the labeling substance. 1. A method for immunologically analyzing a specific component in a fluid sample, wherein the substance is an arylmercury derivative and further contains a compound represented by the following general formula [II] and/or a salt thereof. General formula [II] ▲ Numerical formulas, chemical formulas, tables, etc. are included ▼ [In the formula, R_1 and R_2 represent an alkyl group having 1 to 5 carbon atoms and having a hydroxyl group or a sulfone group as a substituent in the main chain. ] (4) The compound represented by the general formula [II] is piperazine-N,N'-bis(2-ethalsulfonic acid) or N-2
-Hydroxyethylpiperazine-N'-2-ethalsulfonic acid.The method for immunologically analyzing a specific component in a fluid sample according to claim 3. (5) Either a biologically active substance that does not bind to a specific component in a fluid sample or a substance that specifically binds to the biologically active substance, and a substance that specifically binds to a labeling substance and modulates the signal caused by the labeling substance, which are the same or In an immunological analysis element that is separately immobilized on a separate carrier and contained in part or all of the porous reaction layer, the substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance is an aryl. An immunological analytical element which is a mercury derivative and further contains a compound represented by the general formula [I] and/or a salt thereof. (6) The compound represented by the general formula [I] is bis(
6. The immunological analytical element according to claim 5, which is 2-hydroxyethyl)iminotris(hydroxymethyl)methane. (7) Either a biologically active substance that does not bind to a specific component in a fluid sample or a substance that specifically binds to the biologically active substance, and a substance that specifically binds to a labeling substance and modulates the signal caused by the labeling substance. are separately immobilized on the same or separate carriers to specifically bind to the labeling substance and modulate the signal caused by the labeling substance in the immunological analysis element contained in part or all of the porous reaction layer. An immunological analytical element characterized in that the substance is an arylmercury derivative and further contains a compound represented by the general formula [II] and/or a salt thereof. (8) The compound represented by the general formula [II] is piperazine-N,N'-hydroxyethylpiperazine-N'-2-
The immunological analytical element according to claim 7, which is etalsulfonic acid. (9) In the analysis process of a specific component in a fluid sample, a.
either a label formed by binding a labeling substance to the specific component or its analogue, or a label formed by binding a labeling substance to a substance that specifically binds to the specific component; b. at least one type of carrier; The above-described analysis system uses a combination of a porous reaction layer immobilized with a substance that specifically binds to the specific component and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance. In the method for analyzing a specific component, the substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance is an arylmercury derivative, and further comprises a compound represented by the general formula [I] and/or a salt thereof. An immunological analysis method for a specific component in a fluid sample, comprising: (10) The method for immunologically analyzing a specific component in a fluid sample according to claim 9, wherein the compound represented by the general formula [I] is bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. (11) In the analysis process of specific components in a fluid sample, a
, a label formed by binding a labeling substance to the specific component or an analog thereof, or a labeling body formed by binding a labeling substance to a substance that specifically binds to the specific component, and b. at least one type of carrier. and a porous reaction layer containing an immobilized substance that specifically binds to the specific component and a substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance. In the method for analyzing a specific component, the substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance is an arylmercury derivative, and further comprises a compound represented by the general formula [II] and/or a salt thereof. An immunological analysis method for a specific component in a fluid sample, comprising: (12) The compound represented by the general formula [II] is piperazine-N,N'-bis(2-ethanesulfonic acid) or N-
12. The method for immunologically analyzing a specific component in a fluid sample according to claim 11, wherein the component is 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. (13) Separately immobilizing a substance that specifically binds to a specific component in a fluid sample and a substance that specifically binds to a labeling substance and modulating a signal caused by the labeling substance on the same or separate carriers,
In the immunological analysis element contained in part or all of the porous reaction layer, the substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance is an arylmercury derivative, and further has the general formula [ An immunological analytical element comprising a compound represented by [I] and/or a salt thereof. (14) The immunological analytical element according to claim (13), wherein the compound represented by the general formula [I] is bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. (15) A substance that specifically binds to a specific component in a fluid sample and a substance that specifically binds to a labeling substance and modulates a signal caused by the labeling substance are separately immobilized on the same or separate carriers to form a porous reaction. In the immunological analysis element contained in part or all of the layer, the substance that specifically binds to the labeling substance and modulates the signal caused by the labeling substance is an arylmercury derivative, and further has the general formula [II] Compounds shown and/or
or a salt thereof. (16) The compound represented by the general formula [II] is piperazine-N,N'-bis(2-ethanesulfonic acid) or N-
The immunological analytical element according to claim 15, which is 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.