JPH0949800A - Composition for detecting reaction and method for using the composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To judge presence or absence of reaction occurrence in an early stage and presence or absence of a material to be detected by the naked eye observation with a superior observation property by using a critical point of an adoption pH value of a buffer solution and a pH indicator. SOLUTION: A pH indicator and a component for detecting reaction consisting of a buffer agent that loses a buffer function in the vicinity of a boundary pH in a region of the pH indicator are utilized. A condition wherein the first pH of a reaction system corresponds to a value in the vicinity of a boundary value in an adaptive pH region of a buffer solution is selected. As a result, when the slight variation of a hydrogen ion concentration caused by the reaction occurs so as to shift toward the outside of the pH region, the buffer solution that has so far had a function of suppressing the variation of the pH until the time point loses the buffer operation characteristic by virtue of the shift of the buffer solution to the outside of the adaptive pH region so that the pH of the reaction system suddenly changes. Therefore, it is possible to detect whether or not the reaction advances to the desired extent with high accuracy and readily by the naked eye by combining the pH indicator that corresponds to the change point therewith.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reaction detection composition for facilitating macroscopic observation of a pH change of a reaction solution caused by an enzymatic reaction or the like by using a critical point of an applied pH value of a buffer solution and a pH indicator. It is related to its use.

[0002]

2. Description of the Related Art In the detection of various compounds by enzyme immunoassay, enzyme method, etc., a color developing solution is used which causes a continuous and quantitative change in color depending on the reaction. The applicant also previously filed Japanese Patent Application No. 3-183294, 3-1.
85189, 3-185192, 4-275489.
No. 5-230868, 5-251067, 5-3
40553, 6-332475, 6-338891.
No. and reference number HP001721-1 are proposed.

Generally, in the colorimetric analysis using the enzyme immunoassay method or the enzyme method, the amount of a specific substance is measured by utilizing the fact that the color change due to the reaction occurs continuously and quantitatively. ing. For example, the Boehringer Mannheim product name F kit measures the color change of the coenzyme associated with the decomposition reaction by decomposing a substance to be detected (such as lactic acid or malic acid) contained in foods with a specific enzyme. By doing so, the substance to be detected is quantified. The Bonal Kit, a trade name manufactured by Dojindo Laboratories, is used to quantify the substances to be detected (for example, iron ions and chromium ions) in wastewater by measuring the color change of the chelate using the chelate reaction. is there.

Most of such quantitative reagent kits require instruments such as a spectrophotometer and a colorimeter, color samples, and the like in order to measure or identify color changes.

When using a device, the place of measurement is limited to the place where the device is installed, and therefore, for example, environmental pollutants cannot be measured at the site of contamination. For this reason, it is necessary to take the substance to be measured back to the place where the device is installed after collecting the substance to be detected, which is unsuitable when the substance to be detected changes over time.

When the measurement or judgment is visually performed as compared with a color sample, an error occurs in the measurement or judgment due to observation conditions such as illumination and individual differences. For example, when measuring environmental pollutants or the like at a polluted site, it is sufficient to determine whether or not a substance to be detected is present or below a regulation value, and it is important to be able to immediately measure or determine at the site. Further, it is necessary to avoid making an error in measurement or judgment due to observation conditions or individual differences.

[0007]

It is an object of the present invention to have an excellent recognizability and to judge by visual observation whether or not a desired reaction occurs, the presence or absence of a substance to be detected or whether their concentration exceeds a reference value. The point is to provide a composition for facilitating reaction detection.

[0008]

The present inventors have found that the line a in FIG.
As shown in, when the reaction progresses quantitatively, the change in pH changes at a constant rate over time, so even if an attempt is made to detect that the reaction has reached a predetermined stage with a pH indicator, the degree of color development will be high. It is difficult to detect accurately because the change in pH is gentle, but if the pH value can be changed as shown by the line b in FIG. 5, a change in the degree of color development due to the pH indicator will appear remarkably, so accurate detection is possible. That is, the present invention has been completed.

That is, the key point of the present invention is that the property of the buffer solution is used to make the change shown by the line b in FIG. The buffer solution has an action of absorbing changes in hydrogen ion concentration, and as shown in Table 1 below, each buffer solution has an applicable pH range in which the pH of the reaction solution can be kept constant. The buffer is used in the pH range within this range. On the other hand, in the present invention, by selecting a state in which the initial pH of the reaction system corresponds to a value in the vicinity of the boundary value of the pH range where the buffer solution is applied, the concentration of minute hydrogen ion concentration generated by the reaction can be reduced. When a change occurs outside the pH range, the buffer solution that had the function of suppressing the pH change up to that point deviates from the applicable pH range, causing the buffer solution to lose its buffering effect and react. The pH of the system changes all at once.
Therefore, by combining with a pH indicator that matches this change point, whether or not the reaction has proceeded to a desired degree can be detected very accurately and easily with the naked eye.

The first aspect of the present invention relates to a reaction-detecting composition comprising a pH indicator and a buffering agent that loses its buffering function in the vicinity of the boundary pH of the discolored pH region of the pH indicator.

The second aspect of the present invention is that, as the reaction progresses, the pH
The composition for reaction detection according to claim 1 is added to a reaction system in which the pH changes, and the pH of the reaction system is adjusted to a pH at which the buffer function of the added buffer can be fully exerted. The present invention relates to a detection method for detecting a pH change of a reaction solution by a sharp color change of a pH indicator.

The third aspect of the present invention is that the reaction system (a) is of the general formula (I) capable of recognizing a monoclonal antibody which shows a specific immune reaction to methamphetamine.

Embedded image (However, n is an integer of 0, 1, 2, 3 or 4,
R is a hydrogen or an alkyl group), and shows a specific immune reaction to (b) methamphetamine on a carrier carrying a complex of an amine and a supporting protein selected from the group consisting of compounds represented by An enzyme-labeled monoclonal antibody obtained by labeling a monoclonal antibody with an enzyme and (c) methamphetamine in a measurement sample are allowed to undergo a competitive reaction, and after the reaction is completed, the enzyme-labeled monoclonal bound to the carrier of (a) above. The detection method according to claim 2, which is a reaction system in which (d) a substrate is reacted with the antibody.

A fourth aspect of the present invention is the following general formula (I) which allows the reaction system to recognize a monoclonal antibody (A) which shows a specific immune reaction to methamphetamine.

Embedded image (However, n is an integer of 0, 1, 2, 3 or 4,
R is hydrogen or an alkyl group) An antigen-enzyme complex for detecting methamphetamine comprising a complex of an amine and an enzyme selected from compounds represented by the formula (B) a monoclonal antibody showing a specific immunoreactivity to methamphetamine The detection method according to claim 2, which is a reaction system in which a carrier carrying an antibody, (C) a methamphetamine in a measurement sample, and (D) a substrate are mixed and reacted.

The fifth aspect of the present invention is: (i) an enzyme-labeled monoclonal antibody-containing container obtained by labeling a monoclonal antibody which shows a specific immune reaction to methamphetamine with an enzyme, and (ii) methamphetamine Of the general formula (I) capable of recognizing a monoclonal antibody showing a specific immune reaction

[Chemical 6] (However, n is an integer of 0, 1, 2, 3 or 4,
R is hydrogen or an alkyl group), a carrier carrying a complex of an amine and a supporting protein selected from the group consisting of compounds represented by: (iii) a substrate solution-containing container, (iv)
The reaction-detecting composition-containing container according to claim 1, (v) pH
The present invention relates to a kit for detecting methamphetamine, which comprises a container containing an adjusting reagent.

A sixth aspect of the present invention is (i) a container containing an antigen-enzyme complex for detecting methamphetamine according to claim 5, (ii)
A carrier carrying a monoclonal antibody showing a specific immune reaction to methamphetamine, (iii) a substrate solution-containing container, (iv) a reaction-detecting composition-containing container according to claim 1, (v) a pH adjusting reagent The present invention relates to a methamphetamine detection kit including a containing container.

The buffer solution is, for example, Funakoshi Co., Ltd. General Catalog, 1994-95, No. 1876-1881.
List of buffer properties on page, published by Maruzen Co., Ltd. January 30, 1984, Nobumasa Imura, 5 members, "Biochemistry Handbook"
Pages 474-484, Boehringer Mannheim, Inc., Research Reagent Catalog, 1994-95, 273-
275, September 25, 1966, Maruzen Co., Ltd., Chemistry Handbook, Basic Edition, pages 1312 to 1320, and the like, and various buffer solutions described therein can be used. The concentration of the buffer solution to be added is preferably dilute, and particularly preferably 0.1-1.0 mM.

The following table shows representative buffer solutions and their pH ranges.

[0018]

[Table 1]

Note 1. MES: 2-morpholinoethanesulfonic acid monohydrate, 1. Bis-Tris: bis (2-hydroxyethyl)
Iminotris (hydroxymethyl) methane 3. ADA: N- (2-acetamido) iminodiacetic acid 4. PIPES: Piperazine-1,4-bis (2
-Ethanesulfonic acid) 5. ACES: N- (2-acetamido) 2-aminoethanesulfonic acid 6. MOPSO: 2-hydroxy-3-morpholinopropanesulfonic acid 7. BES: N, N-bis (2-hydroxyethyl) -2-aminoethane-sulfonic acid 8. MOPS: 3-morpholinopropanesulfonic acid 9. TES: N-tris (hydroxymethyl) methyl-2-amino-ethanesulfonic acid 10. HEPES: 2- [4- (2-hydroxyethyl) -1-piperazinyl] -ethanesulfonic acid 11. DIPSO: 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid 12. TAPSO: 2-hydroxy-N-tris (hydroxymethyl) -methyl-3-aminopropanesulfonic acid 13. POPSO: piperazine-1,4-bis (2
-Hydroxy-3-propanesulfonic acid dihydrate 14. HEPPSO: 2-hydroxy-3- [4-
(2-Hydroxyethyl) -1-t'perazinyl] propanesulfonic acid monohydrate 15. EPPS: 3- [4- (2-hydroxyethyl) -1-piparadinyl] propanesulfonic acid 16. Tricine: N- [tris (hydroxymethyl) methyl] glycine 17. Bicine: N, N-bis (2-hydroxyethyl) glycine 18. TAPS: N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid 19. CHES: N-cyclohexyl-2-aminoethanesulfonic acid 20. CAPSO: N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid 21. CAPS: N-cyclohexyl-3-aminopropanesulfonic acid

Examples of pH indicators include, for example, September 1966.
March 25, published by Maruzen Co., Ltd., Chemistry Handbook Basic Edition, 130
Pages 5 to 1308, Funakoshi Co., Ltd. General Catalog, 19
94-95, p.
It is possible to use an H indicator.

Specific examples of the pH indicator are shown in Tables 2 and 3 below.

[0022]

[Table 2] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Product name pH range ━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Congo red 3.0-5.0 ━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━ methyl orange 3.2-4.5 ━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━ Resazurin 3.8-6.5 ━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━ Methyl red, neutral 4.5-6.0 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━ Bromocresol purple 5.2-6.8 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━ chlorophenol red 5.2-6.8 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Brilliant yellow 6.4-9.4 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Neutral red 6.8-8. 0 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Phenol red (water-soluble) 6.8-8.2 ━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ m-cresol red 7.0-8.8 ━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ m-cresol red sodium acid (water-soluble) 7.0-8.8 ━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━ m-cresol purple (water-soluble) 7.5-9.0 ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━

[Table 3] ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ Product name pH range ━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━━━━━━━ thymol blue 8.0-9.0 ━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━━━ thymol blue (water-soluble) 8.0-9.0 ━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━━━━━━━ Phenol violet 8.0-10.0 ━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━━ Nile Blue A 9.5-10.5 ━━━━━━━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━ Alizarin Yellow R 9.6-12.0 ━━━━━━━━━━━━━━━━━━━━━ ━━━━━━━━━━━━━ The initial pH of the reaction system is the application of the buffer solution to be added As a method for adjusting the boundary value of the pH, it is common to adjust the pH while measuring it. However, it is desirable to add a pH indicator to examine the relationship between the hydrogen ion concentration and the color development of the pH indicator, and to adjust based on this result.

The composition for detecting a reaction of the present invention is useful as a method for ascertaining the progress of various reactions accompanied by a pH change or as a method for detecting a specific substance accompanied by a pH change. A method that applies an oxidation reaction or a decomposition reaction of a specific substance using an enzyme, an immunological method that uses an enzyme that catalyzes the oxidation reaction or a decomposition reaction of a specific substance, and a catalyst that oxidizes or decomposes a specific substance Examples thereof include methods for detecting microorganisms or diseases using enzyme activity as an index.

As a method applying the oxidation reaction or decomposition reaction of a specific substance using the enzyme, various dehydrogenases such as an alcohol detection method using alcohol dehydrogenase and a galactose detection method using galactose dehydrogenase were used. Methods, methods using various oxidases such as glucose detection method using glucose oxidase and bilirubin oxidase detection method, aspartic acid detection method using aspartate ammonium lyase, phenylalanine ammonia lyase method Methods using various amino acid ammonia lyases such as phenylalanine detection methods, detection methods for triglycerides using various lipases, detection methods for esters using various esterases, detection of urea using urease Or the like can be mentioned law. There are also reactions that combine these reactions. For details, refer to "Enzyme Agents-Basic and Actual-" (Takeshi Tanimoto, Hirokawa Shoten, 1991) and the Boehringer Mannheim Co., Ltd. research reagent catalog 1994'95. It is described in the literature.

As an immunological method using an enzyme that catalyzes the oxidation reaction or decomposition reaction of the specific substance, various methods in which an antibody or an antigen is labeled with the enzyme have been proposed. As the enzyme used for labeling, various dehydrogenases such as alcohol dehydrogenase and galactose dehydrogenase, various oxidases such as glucose oxidase and bilirubin oxidase, various amino acid ammonia lyases such as aspartate ammonium lyase and phenylalanine ammonia lyase, various lipases, Esterase, urease and the like can be mentioned.

As a method for detecting a microorganism or disease using the enzyme activity that catalyzes the oxidation reaction or decomposition reaction of the specific substance as an index, a method for detecting a lactic acid bacteria group using the production of lactic acid as an index, or an acid production from lactose is used. Detection method of intestinal bacterial group using index, detection method of Helicobacter pylori using urease activity, adenosine deaminase and β
-A method for detecting inborn errors of metabolism using galactosidase as an index can be mentioned.

As an example of the reaction system, the reaction system for detecting methamphetamine described above can be mentioned first, but the following reaction system is also available.

Example of reaction (1) A reaction in which ammonia combines with α-oxoglutaric acid to form L-glutamic acid

[Chemical 7] Note) GIDH: Glutamate dehydrolog
enase (glutamate dehydrogenase) NADH: nicotinamide dinucleotide (reduced type) NAD ⁺ : nicotinamide dinucleotide (oxidized type) (2) Reaction of converting ethanol into acetic acid

Embedded image Note) ADH: Alcohol dehydrogenase AIDH: Aldehyde dehydrogenase (3) Reaction to convert glucose to gluconate-6-phosphate

Embedded image Note) ATP: adenosine 3-phosphate HK: hexose kinase ADP: adenosine 2-phosphate G-6-P: glucose 6-phosphate NADP: nicotinamide dinucleotide phosphate NADPH: nicotinamide dinucleotide phosphate (reduced form) ) (4) Reaction of converting glucose, fructose and mannose into gluconic acid-6-phosphate

Embedded image Note) Gluconic acid-6-P: Gluconic acid-6-phosphate (5) Reaction to convert glycerol to pyruvic acid or lactic acid

Embedded image Note) GK: Glycerol kinase Glycerol-3-P: Glycerol 3-phosphate PEP: Phosphoalepyruvic acid PK: Pyruvate kinase NADH: Nicotinamide dinucleotide (reduced form) LDH: Lactate dehydrogenase (6) Lactose (lactose), Reaction to convert β-galactose to galactonic acid

[Chemical 12] Note) Gal DH: Galactose dehydrogenase (7) Reaction to convert lactose and glucose into gluconic acid-6-phosphate

Embedded image (8) Reaction of converting lecithin into pyruvic acid or lactate

Embedded image (9) Reaction of converting maltose, sucrose and glucose into gluconic acid-6-phosphate

Embedded image Note) G-6-P-DH: Glucose 6-phosphate dehydrogenase (10) Reaction to convert raffinose to galactonolactone (which becomes lactone acid immediately in water)

Embedded image (11) Reaction of converting starch to gluconic acid-6-phosphate

Embedded image Note) G-6-P ・ DH: Glucose 6-phosphate dehydrogenase (12) Reaction to convert saccharose to gluconic acid via glucose

Embedded image (13) Reaction for converting sucrose, glucose and fructose into glucose and 6-phosphate

Embedded image (14) Reaction of converting urea and ammonia into L-glutamic acid

Embedded image

Typical amines represented by the general formula (I) are compounds represented by the following formulas (II) to (V) or N-methylated compounds thereof. The following formula (II)

[Chemical 21] A phenethylamine represented by the following formula (III)

Embedded image N-methylphenethylamine represented by the following formula (IV)

Embedded image 3-phenylpropylamine represented by the following formula (V)

Embedded image 4-phenylbutylamine represented by

Examples of the enzyme include β-galactosidase, glucose oxidase, acetylcholinesterase, alkaline phosphatase, horseradish peroxidase, lactate dehydrogenase, glucoamylase,
Examples thereof include tyrosinase and urease. Examples of the reagent for binding the amine represented by the general formula (I) to the enzyme include glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide and the like. Used. These amines represented by the above general formula (I) may be directly bound to the enzyme, but it is preferable to carry out aminoalkylation or carboxyalkylation using a suitable reagent and then bind to the enzyme.

Examples of suitable combinations of these enzymes and amines are shown in Table 4 below.

[Table 4] Reference (1) Antibodies Volume II, D Catty, Ed. (IRL PRESS) P97-154 (2) Biochemical Experimental Method 27. Enzyme labeling method, Eiji Ishikawa, Academic Publishing Center P5-29

The carrier is preferably a container such as a test tube or pipette, or a stick, but in addition to this, any shape such as a sphere, a cube or a needle can be used, and the material is There is no particular limitation, but those having a property of easily attaching proteins are suitable, and synthetic resins (for example, polystyrene, polyethylene, polypropylene, polymethylmethacrylate, polyamide, polyester, etc.), rubber, glass, silica, nitrocellulose, Acetyl cellulose etc. can be mentioned. In the case of a container, it is supported by forming a layer of the complex or a layer containing the same on the inner wall thereof in the case of a stick or the like on the surface thereof.

A. Preparation of immunizing antigen Methamphetamine was bound to an arbitrary protein to obtain a complex, which was used as an immunizing antigen. Examples of the protein include bovine serum albumin, ovalbumin, Jinkasai hemocyanin, thyroglobulin, γ-globulin and the like. Further, as a reagent for binding methamphetamine to a protein, glutaraldehyde, 1-ethyl-3
-(3-Dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide and the like are used. Methamphetamine may be bound to the protein as it is, but it is preferable to bind to the protein after aminoalkylation or carboxyalkylation using a suitable reagent.

B. Preparation of lymphocytes The immunizing antigen is administered to a mammal (eg, mouse, rat, etc.) 4 to 12 times every other week for 4 to 12 times into the abdominal cavity, subcutaneously or directly into the spleen, and sufficient antibody against the antigen is produced. After confirmation, lymphocytes are obtained from the blood, lymph nodes, spleen, etc. of the animal. At this time, it is preferable to administer an adjuvant (including alum, killed tuberculosis bacteria, nucleic acid, etc.) as an immunostimulant together with the antigenic substance to the animal as an emulsion. Examples of means for confirming the production of antibodies include means for collecting venous blood from the immunized animal and using the enzyme immunoassay in the section of hybridoma cell selection (C.) described below.

C. Cell Fusion and Selection of Hybridoma Strain As the myeloma cells used for cell fusion, for example, mouse-derived P3-X63Ag8-U1 strain, SP2 / 0-A
g14 strain, P3-NS1 / 1-Ag4.1 strain, P3-X
63-Ag8.653 strain, P3-X63-Ag8 strain, M
PC-11 strain, 210 strain derived from rat, RCY3 strain, A
G1, 2, 3 strains, human-derived SKO-007 strain, GH1
5006TG-A12 strain etc. can be mentioned. In the cell fusion, the lymphocytes and the myeloma cells of the immunized animal as described above are mixed at a ratio of about 2: 1 to 10: 1, and the mixture is centrifuged to obtain a mixed precipitate of the lymphocytes and the myeloma cells. It can be obtained by adding a cell culture medium containing polyethylene glycol or Sendai virus to the suspension and suspending it.
It is preferable to use 0% to 60% polyethylene glycol.

Hybridoma strains are selected by centrifuging the cell suspension after fusion to remove the supernatant, and adding hypoxanthine,
It can be carried out by resuspending in a cell culture medium containing aminopterin, thymidine and 10% to 20% fetal bovine serum, and dispensing this suspension into a culture plate. The hybridoma cells selected by this operation are further cultured in a cell culture medium containing hypoxanthine, thymidine, and 10% to 20% fetal bovine serum, and finally 1
Culture in cell culture medium containing 0-20% fetal calf serum. Since the hybridoma cells that have proliferated produce antibodies in the culture medium supernatant, the presence or absence of the desired antibody can be examined using oxygen immunoassay, radioisotope immunoassay, plaque assay, agglutination method, etc. It is desirable to use an enzyme immunoassay.

This enzyme immunoassay can be carried out as follows. A. A fixed amount of the immunizing antigen prepared in 1. is dispensed into each well of a 96-well plate for enzyme immunoassay. The 96-well plate for enzyme immunoassay is allowed to stand for a certain period of time to immobilize the immunizing antigen in the well. After a certain period of time, the contents are discarded, and then a protein solution such as bovine serum albumin, ovalbumin, Jinkasai hemocyanin, skim milk, and immunoglobulin is dispensed into each well in a fixed amount. This is to prevent non-specific adsorption of the antibody produced by the hybridoma cells to the inside of the well during the next operation. A fixed amount of the hybridoma cell culture supernatant is added to each well of the 96-well plate for enzyme immunoassay after the above operation.
Each well is washed after standing for a certain period of time, and then an enzyme-labeled antibody (for example, when mouse is used, an enzyme such as alkaline phosphatase, horseradish peroxidase, or β-galactosidase is bound to an anti-mouse immunoglobulin G antibody) ) Is added to each well in a fixed amount. After standing for a certain period of time, each well is washed and a substrate solution corresponding to the enzyme used is added to carry out an enzyme reaction. When the target antibody is present in the culture supernatant, the color change of the substrate caused by the enzymatic reaction can be visually or mechanically confirmed. In this way, hybridoma cells producing the desired antibody can be obtained.

The monoclonal antibody showing a specific immune reaction to methamphetamine is hybridoma 8C1.
It is preferable that the antibody is produced by a strain (Microtech Lab. No. 13148).

The 8C1 strain was methamphetamine (MA in the table).
(Shown in)). Its excellent selectivity is shown in Table 5.

[0040]

[Table 5] In the table, MA, methamphetamine DBED, N-N'-dibenzylethylenediamine A, amphetamine MPA, methoxyphenamine NOR-EP (+), norephedrine (+) OH-MA, p-hydroxymethamphetamine OH. -NOR-EP, p-hydroxynorephedrine OH-EP, p-hydroxyephedrine OH-A, p-hydroxyamphetamine ME, methylephedrine NOR-EP-(-), norephedrine (-) EP is , Ephedrine MES is Mescaline. From this, it was found that the monoclonal antibody produced by the hybridoma cell line 8C1 has low reactivity with other similar compounds other than MA and is suitable for the measurement of MA. The anti-monoclonal antibody produced by the hybridoma cell line 8C1 is an antibody having a λ type light chain and a γ1 type heavy chain.

D. Cloning of hybridoma cells Since the cell population in the well confirmed in 1. may be a mixture of genetically different hybridoma cells, it is necessary to obtain a hybridoma cell group consisting of a single gene by a cloning operation. Examples of this method include a limiting dilution method, a single cell manipulation method, and a method of picking up colonies on soft agar one by one. It is preferable to use the limiting dilution method because no special device is used. This limiting dilution method can be performed as follows. The above hybridoma cells were adjusted to an appropriate concentration with a cell culture medium containing 0 to 20% fetal bovine serum, and the cell suspensions adjusted to each concentration were fixed in each well of an enzyme immunoassay 96-well plate. Dispense the amount.
The hybridoma cells are cultured in a CO ₂ incubator, and wells in which it is confirmed that one colony is derived from one hybridoma cell are selected. Whether the monoclonal antibody present in the supernatant of these wells recognizes methamphetamine was determined as described in the above C. Re-examine by the enzyme immunoassay method of. When a group of hybridoma cells consisting of a single gene is obtained in this manner, it can be said that the antibody produced from this cell is a monoclonal antibody.

E. Preparation of Monoclonal Antibody The preparation of the monoclonal antibody against methamphetamine is described in D. In the flask, the hybridoma cells obtained in
Alternatively, it can be performed by culturing in the abdominal cavity of a mammal. Culturing the hybridoma cells in the flask is performed using a cell culture medium containing 0 to 20% fetal bovine serum. At this time, the hybridoma cells are allowed to grow to the maximum, and then centrifuged to obtain the secreted monoclonal antibody in the supernatant. The target monoclonal antibody can be separated and purified by operations such as salting out, dialysis, ion exchange chromatography, affinity chromatography and the like. For the culture of hybridoma cells in the abdominal cavity of animals, 1 × 10 ⁶ to 10 × 10 ⁸ cells are injected into the abdominal cavity of animals to which mineral oil such as pristane has been administered. As the animal species used at this time, it is desirable to use an animal of the same species and strain as the myeloma cells used for producing the hybridoma cells, so that the hybridoma cells can easily proliferate. For example, when using a mouse, this operation
After 2 weeks, proliferation of hybridoma cells is observed in the abdominal cavity, and ascites accumulates in the abdominal cavity. Since the target monoclonal antibody exists in ascites, the ascites can be collected from the abdominal cavity, and the monoclonal antibody can be separated and purified by operations such as salting out, dialysis, ion exchange chromatography, and affinity chromatography.

F. Preparation of urease-labeled monoclonal antibody The purified monoclonal antibody and urease were treated in a buffer to obtain an enzyme-labeled monoclonal antibody. The treatment for binding the antibody and urease can be performed by using a cross-linking agent such as glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleinimidobenzoyloxysuccinimide. . As the buffer solution, phosphoric acid, Tris-hydrochloric acid, sodium acetate, sodium carbonate or the like can be used, and the pH is preferably around neutral.

G. Immobilization of MA-supporting protein complex on carrier (1) Synthesis of MA-supporting protein complex Aminobutylated or carboxymethylated methamphetamine was prepared, and any supporting protein was bound to this to obtain a complex. Examples of the supporting protein include bovine serum albumin, ovalbumin, mussel hemocyanin, thyroglobulin, γ-globulin and the like. Also,
As a cross-linking agent, glutaraldehyde, 1-ethyl-3
-(3-Dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide and the like are used.

(2) Immobilization of MA-supported protein complex on carrier Since synthetic resins such as polystyrene, polyethylene, polypropylene, polymethylmethacrylate, and styrene adsorb proteins, they are used as containers or sticks made of these materials. The MA-supporting protein complex can be efficiently bound to the surface of the carrier. This reaction is at room temperature,
Although it is completed in 5 to 15 minutes, after the reaction is completed, by using a cross-linking agent such as glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleinimidobenzoyloxysuccinimide, It can be adjusted so that the complex or the monoclonal antibody is not separated from the carrier, and more complex is adsorbed on the carrier.

H. MA measuring method using urease-labeled monoclonal antibody In the MA measuring method of the present invention, G. MA- adjusted in (2)
A support protein complex-binding carrier (hereinafter abbreviated as MA support carrier) is prepared by using 1 to 100 μg / ml of a urease-labeled monoclonal antibody solution and 0.1 to 2% of a protein such as bovine serum albumin (BSA) or Kasagai hemocyanin ( KL
H) and ovalbumin (OVA) in a buffer containing a biopolymer such as urine or saliva. If MA is not present in the sample (negative), the urease-labeled monoclonal antibody will react with MA by the antigen-antibody reaction.
-Binds to a support carrier. This carrier is washed with a washing solution (phosphate, Tris-HCl buffer, sometimes containing 0.01 to 1% surfactant), and then a substrate corresponding to the labeled urease, that is, a urea solution. Let it react in
The change is confirmed with the naked eye or an analyzer by the degree of color development. In this reaction, when MA is present in the sample (positive), the urease-labeled monoclonal antibody in the reaction solution binds to MA, and the binding to the MA support carrier is inhibited. Therefore, the conversion of the substrate is also inhibited, and the presence of MA in the sample in the positive case can be confirmed as compared with the negative case in which the substrate is sufficiently converted. That is, if MA was not present in the sample, the urease-labeled monoclonal antibody would react with the MA-supporting protein complex,
As a result, urea is decomposed to generate ammonia. Therefore, the pH of the reaction system rises, and this is detected by a pH indicator (including pH reagent paper). MA in the sample
In the presence of a, the body cannot bind to the MA-support and therefore cannot decompose most of the urea as MA reacts with the antibody. As a result, the pH of the system hardly rises and the discoloration of the pH indicator does not occur.

I. Support of monoclonal antibody on carrier Since synthetic resins such as polystyrene, polyethylene, polypropylene, polymethylmethacrylate, and styrene adsorb proteins, the monoclonal antibody can be efficiently deposited on the carrier surface of containers or sticks made of these materials. Can be combined. This reaction is completed in 5 to 15 minutes at room temperature, but after the reaction is completed, a crosslinking agent such as glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide is used. By doing so, it can be adjusted so that the monoclonal antibody is not peeled off from the carrier, and more monoclonal antibody is adsorbed on the carrier.

J. Selection of Amines Recognized by Monoclonal Antibody that Shows Specific Immune Reaction to Methamphetamine The antigen solution for immunization prepared in the above (A) is dispensed into each well of a 96-well plate for enzyme immunoassay in a fixed amount. The 96-well plate for enzyme immunoassay is allowed to stand for a certain period of time to immobilize the immunizing antisource in the well. After a certain time, discard the contents,
Next, a fixed amount of a protein solution such as bovine serum albumin, ovalbumin, Jinkasai hemocyanin, skim milk, immunoglobulin is dispensed into each well. This is to prevent the antibody produced by the hybridoma cells from nonspecifically adsorbing inside the well during the next operation. One of various amines including the amines represented by the general formula (I) and the monoclonal antibody purified by the method (D) in each well of the 96-well plate for enzyme immunoassay after the above operation And mix and dispense a fixed amount. Next, an enzyme-labeled antibody (for example, in the case of using a mouse, an anti-mouse immunoglobulin G antibody to which an enzyme such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, etc. is bound) is added in a fixed amount to each well. After standing for a certain period of time, each well is washed and a substrate solution corresponding to the enzyme used is added to carry out an enzyme reaction. When the target antibody is present in the culture supernatant, the color change of the substrate caused by the enzymatic reaction can be visually or mechanically confirmed. If a compound that can be recognized by this monoclonal antibody exists in the amines used in the experiment, the compound binds to the monoclonal antibody and the antigen carried in each well of the 96-well enzyme immunoassay plate is carried. Competitively inhibits the binding between the antibody and the monoclonal antibody. Therefore, since the monoclonal antibody does not remain in the well in which the mixture of the compound that can be recognized by the monoclonal antibody and the monoclonal antibody is dispensed, the color change of the substrate that should occur due to the enzymatic reaction is weakened or not observed. In this way, a compound that can be recognized by the monoclonal antibody can be selected from various amines.

K. Preparation of Antigen-Enzyme Complex A complex in which the amines represented by the general formula (I) and an arbitrary enzyme were bound was synthesized and used as an antigen-enzyme complex. As the enzyme, β-galactosidase, glucose oxidase, acetylcholinesterase, alkaline phosphatase, horseradish peroxidase, lactate dehydrogenase, glucoamylase, tyrosinase,
Urease or the like is used. As the reagent for binding the enzyme represented by the general formula (I) to the enzyme, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide and the like are used. To be

L. Methamphetamine detection method using antigen-enzyme complex In the methamphetamine detection method of the present invention, a buffer solution containing 0.1 to 2% of a protein (eg, bovine serum albumin, Jinkasai hemocyanin, ovalbumin) as a monoclonal antibody-binding carrier. Inside, react with samples such as urine and saliva. If methamphetamine is not present in the sample (negative), nothing binds to the monoclonal antibody-bound carrier. This carrier is washed with a suitable washing solution and reacted with the antigen-enzyme complex. The antigen-enzyme complex binds to the monoclonal antibody-binding carrier by the antigen-antibody reaction. Furthermore, this carrier is washed with an appropriate washing solution, and a substrate solution corresponding to each enzyme of the antigen-enzyme complex is reacted,
Confirm the change visually or mechanically. In this reaction, when methamphetamine is present in the sample (positive), methamphetamine binds to the monoclonal antibody-bound carrier, and the binding of the next antigen-enzyme complex to the monoclonal antibody-bound carrier is inhibited. . Therefore, the presence of methamphetamine in the sample in the positive case can be confirmed as compared with the negative case in which the substrate is sufficiently converted by the antigen-enzyme complex.

[0051]

EXAMPLES Examples of the present invention will be specifically described below. It should be noted that these examples are for illustrating the present invention and do not limit the scope of the present invention.

Example 1: Detection of urease activity In order to prepare a reaction-sensing composition for detecting urease activity, it was examined where to set the initial pH of the reaction system. In the reaction detection composition, HEPES buffer (see Table 1 above) was used as a dilute buffer, bromocresol purple was used as a pH indicator, and urea 1 g / liter was used as a substrate. The initial pH of the reaction detection composition is set to 3.
The concentration was changed from 8 to 4.7, and various concentrations of urease were added, and the change in the absorbance at 600 nm accompanying the reaction was measured.
The results are shown in FIGS. 1 and 2. When the initial pH of the reaction detection composition was adjusted to 3.8, 4.0, 4.3, 4.5 and 4.7, the added urease concentration was 2.
The reaction hardly progressed at 5 μg / ml or less, and urease activity could not be recognized with the naked eye, but if there is a certain concentration or more, for example, 5 μg / ml of urease, it is easy to visually judge it. It was

Example 2 Detection of Methamphetamine Using Urease-Labeled Antibody A dilute buffer solution added to the reaction-sensing composition to prepare a reaction-sensing composition for detection of methamphetamine using a urease-labeled antibody. The concentration of the HEPES buffer solution (see Table 1) was examined. In the composition for detecting reaction, bromocresol purple was used as a pH indicator and urea 1 g / liter was used as a substrate. The concentration of the HEPES buffer solution of the reaction detection composition is 0, 0.25 mM,
The concentration was changed to 0.5 mM and 1.0 mM, and various concentrations of methamphetamine were measured using a urease-labeled antibody.
The results are shown in Fig. 3. HEPE of reaction detection composition
When the concentration of the S buffer was adjusted to 0.25 mM, the difference in absorbance at 595 nm between the methamphetamine-added sample (1 μg / ml) and the non-added sample was the largest, and it was possible to visually judge.

Example 3 Detection of Methamphetamine Using Urease-Labeled Antibody In order to prepare the reaction color-developing solution for detection of methamphetamine using urease-labeled antibody, the initial pH of the reaction color-developing solution was examined. The reaction color solution contains H as a dilute buffer.
EPES buffer, bromocresol purple as a pH indicator, and urea 1 g / l as a substrate were used. The initial pH of the reaction color developing solution was changed to 4.25 to 4.8, and various concentrations of methamphetamine were measured using a urease labeled antibody. The results are shown in Fig. 4. When the initial pH of the reaction color development liquid was adjusted to 4.45 to 4.65, the difference in absorbance at 595 nm between the methamphetamine-added sample (1 μg / ml) and the non-added sample was the largest, and it was possible to make a visual judgment. there were.

[0055]

[Effect] According to the present invention, it is possible to accurately determine whether or not a desired reaction has occurred, the presence or absence of a substance to be detected, and whether or not their concentrations exceed a reference value by visual observation. In particular, the present invention can be effectively used for detecting stimulants such as methamphetamine.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of the initial pH of a reaction solution on color development.

FIG. 2 is a graph showing the effect of urease concentration in the reaction solution on color development.

[Fig. 3] Buffer solution that affects color development (HEPES buffer)
It is a graph which shows the influence of density.

FIG. 4 is a graph showing the influence of the initial pH of the reaction solution on the color development when methamphetamine is detected using a urease-labeled antibody.

FIG. 5 is a model graph showing that, according to the present invention, color development rapidly occurs in a certain range of pH.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // C12N 15/02 C12P 21/08 C12P 21/08 9162-4B C12N 15/00 C (C12P 21 / 08 C12R 1:91)

Claims (9)

[Claims] 1. A composition for reaction detection comprising a pH indicator and a buffering agent that loses its buffering function in the vicinity of the boundary pH of the discolored pH region of the pH indicator. 2. A reaction system whose pH changes with the progress of the reaction, to which the composition for reaction detection according to claim 1 is added in advance so that the buffer function of the buffer to which the pH of the reaction system is added can be exerted to the limit. A detection method in which the pH is adjusted and the pH change of the reaction solution due to the progress of the reaction is detected by a sharp color change of the pH indicator. 3. The detection method according to claim 2, wherein the reaction system is an antigen-antibody reaction system. 4. The general formula (I) wherein the reaction system can recognize (a) a monoclonal antibody which shows a specific immune reaction to methamphetamine. (However, n is an integer of 0, 1, 2, 3 or 4,
R is a hydrogen atom or an alkyl group), and a carrier carrying a complex of an amine and a supporting protein selected from the group consisting of compounds represented by (b) shows a specific immune reaction to methamphetamine. The enzyme-labeled monoclonal antibody obtained by labeling the monoclonal antibody with an enzyme and (c) methamphetamine in the measurement sample are allowed to undergo a competitive reaction, and after the reaction is completed, the enzyme-labeled monoclonal bound to the carrier of (a) above. The detection method according to claim 2, which is a reaction system in which (d) a substrate is reacted with the antibody. 5. The following general formula (I): wherein the reaction system can recognize a monoclonal antibody (A) which shows a specific immune reaction to methamphetamine. (However, n is an integer of 0, 1, 2, 3 or 4,
R is hydrogen or an alkyl group), and a methamphetamine-detecting antigen-enzyme complex comprising a complex of an amine selected from compounds represented by the formula (B) a methamphetamine-specific immunoreaction The detection method according to claim 2, which is a reaction system in which a carrier carrying an antibody and (C) methamphetamine and a (D) substrate in a measurement sample are mixed and reacted. 6. The detection method according to claim 4, wherein the enzyme is urease and the substrate is urea. 7. (i) An enzyme-labeled monoclonal antibody-containing container obtained by labeling with a enzyme a monoclonal antibody that exhibits a specific immune reaction to methamphetamine, and (ii) a specific immunity to methamphetamine. General formula (I) recognizable by a reactive monoclonal antibody (However, n is an integer of 0, 1, 2, 3 or 4,
R is hydrogen or an alkyl group), a carrier carrying a complex of an amine and a supporting protein selected from the group consisting of compounds represented by the formula: (iii) a substrate solution-containing container, (iv) A kit for detecting methamphetamine, which comprises a container containing a composition for detecting a reaction, and (v) a container containing a reagent for adjusting pH. 8. (i) A container containing the antigen-enzyme complex for detecting methamphetamine according to claim 5, (ii) a carrier carrying a monoclonal antibody showing a specific immune reaction to methamphetamine, (iii) a substrate solution A kit for detecting methamphetamine, comprising: a container containing; (iv) a container containing the composition for detecting a reaction according to claim 1; and (v) a container containing a reagent for pH adjustment. 9. The kit for detecting methamphetamine according to claim 7, wherein the enzyme is urease and the substrate is urea.