JPH06261784A - Method for detecting methamphethahamine - Google Patents

Abstract

PURPOSE:To obtain a method of simply detecting MA by change of color of a pH indicator by using a urease labeled monoclonal antibody to be specifically reacted with MA and a portable kit for detecting MA. CONSTITUTION:A hybridoma strain of FERM-P 13,148 and production of a hybridoma strain showing specific immune reaction to methamphethamine. A monoclonal antibody showing specific immune reaction to methamphethamine, production of the monoclonal antibody and method of detecting methamphethamine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting methamphetamine which is a stimulant, a monoclonal antibody used therefor, a strain used for producing the monoclonal antibody, and a portable kit for detecting methamphetamine.

[0002]

2. Description of the Related Art With the increase in incidents related to stimulants,
There is a growing need to recognize the user of methamphetamine (hereinafter sometimes abbreviated as MA) simply, quickly and accurately, and a method and a kit for measuring methamphetamine in a simple, highly sensitive and highly selective manner are desired. . JP-A-1-9
No. 6198 discloses that by culturing a cell line obtained by immunizing an animal, a monoclonal antibody having a very high specific reactivity to methamphetamine (MA), which is a stimulant, is obtained. ing. And
Currently, as a measurement system using a monoclonal antibody that specifically recognizes MA, a competitive assay method based on the ELISA method is commercialized. However, this method requires a special scientific instrument such as a spectrophotometer to judge the result, and requires a special technique to some extent because the operation is complicated.

[0003]

[Objective] An object of the present invention is to provide a method for easily detecting MA by discoloration by a pH indicator using a urease-labeled monoclonal antibody that specifically reacts with MA and a portable MA detection kit. is there.

[0004]

[Structure] The first aspect of the present invention relates to a hybridoma strain of Microorganism Research Institute No. 13148.

The second aspect of the present invention is to inoculate a mammal with a methamphetamine immunizing antigen to obtain lymphocytes having an anti-methamphetamine antibody-producing ability, and to fuse the lymphocytes with myeloma cells. The present invention relates to a method for producing the above hybridoma strain, which comprises culturing cells and producing a monoclonal antibody showing a specific immune reaction to methamphetamine.

The third aspect of the present invention relates to a monoclonal antibody which shows a specific immune response to methamphetamine.

A fourth aspect of the present invention relates to a method for producing the monoclonal antibody, which comprises culturing the hybridoma strain.

In a fifth aspect of the present invention, (a) a carrier supporting a complex of methamphetamine and a supporting protein is used to label (b) a monoclonal antibody showing a specific immunoreaction with methamphetamine with urease. The urease-labeled monoclonal antibody thus obtained and (c) methamphetamine in the measurement sample are allowed to undergo a competitive reaction, and after the reaction is completed, (d) urea is added to the urease-labeled monoclonal antibody bound to the carrier of (a) above. The present invention relates to a method for detecting methamphetamine, which comprises reacting and detecting the produced ammonia component.

The method for detecting the ammonia component is the simplest method by changing the pH of the system, and an appropriate pH indicator can be used. The present invention, by selecting and combining urea as a substrate with a urease-labeled monoclonal antibody, urea is decomposed by urease to generate ammonia in the case where MA is not contained in the sample in the negative,
Since the pH of the system rises, if this is used, even a layperson can easily perform the test due to the discoloration of the pH indicator.
Therefore, utilization of the ammonia generation reaction is one of the main points of the present invention.

Examples of pH indicators (including pH test paper) usable in the present invention include bromocresol purple, bromophenol blue, congo red, methyl orange, resazurin, methyl red, chlorophenol red, hematoxylin (alkaline) and brilliant yellow. , Neutral red, phenol red, m-cresol red, m-cresol red sodium salt,
Examples thereof include m-cresol purple and thymol blue.

The substrate used in the present invention may include urea. Urine or saliva of the subject can be used as the measurement sample.

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 that have the property of easily attaching proteins are suitable. Examples include synthetic resins (for example, polystyrene, polyethylene, polypropylene, polymethylmethacrylate, polyamide, polyester, etc.), rubber, glass, nitrocellulose, etc. be able to.

In the case of a container, a layer of the above-mentioned complex or a layer containing the same is formed on the inner wall of the container, and in the case of a stick or the like, the layer is supported.

A sixth aspect of the present invention is (i) a container containing a urease-labeled monoclonal antibody obtained by labeling a monoclonal antibody showing a specific immune reaction to methamphetamine with urease, and (ii) supporting with methamphetamine. The present invention relates to a portable methamphetamine detection kit containing a carrier supporting a complex with a protein, (iii) a substrate solution-containing container containing urea and a pH indicator, or a substrate solution-containing container containing urea and a pH indicator-containing container.
It is preferable that a quantitative dropper and a sample collection container are included in the kit.

A. Preparation of Immunizing Antigen Methamphetamine (MA) is aminobutylated or carboxymethylated to produce aminobutylated or carboxymethylated methamphetamine, and an arbitrary supporting protein is bound to this to obtain a complex, which is used as an immunizing antigen. did.
Examples of the supporting protein include bovine serum albumin, ovalbumin, mussel (Juniper mussel, watermelon) hemocyanin, thyroglobulin, γ-globulin and the like. Further, as the cross-linking agent for binding the aminobutylated or carboxymethylated methamphetamine and the supporting protein, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dimethylformamide, maleimidobenzoyloxysuccinimide and the like are used. To be

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 ELISA method in the section of hybridoma cell selection 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 (P3-U1), S
P2 / 0-Ag14 (SP-2), P3-NS1 / 1-
Ag4.1 (NS-1), P3-X63-Ag8.65
3 (653), P3-X63-Ag8 (X63), MP
C-11, rat-derived 210, RCY3, AG1,
2, 3 (Y3), human-derived SKO-007, GH15
006TG-A12 etc. can be mentioned.

In the cell fusion, the lymphocytes of the immunized animal and the myeloma cells as described above are mixed at a ratio of about 2 to 10: 1, and the mixture is centrifuged to obtain a mixed precipitate of lymphocytes and myeloma cells. Cell culture medium (RPMI1640, MEM, DMEM) containing polyethylene glycol (PEG) or Sendai virus (HVJ)
Etc.) and suspending it, but from the viewpoint of operation, it is preferable to use 30% to 60% PEG (molecular weight 1000 to 8000).

Hybridoma strains are selected by centrifuging the cell suspension after fusion to remove the supernatant, and adding hypoxanthine,
Aminopterin, thymidine (HAT) and 10% to 20%
It can be carried out by resuspending in a cell culture medium containing fetal calf serum (FCS) 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 (HT) and 10% to 20% fetal calf serum (FCS), and finally 10% to 20%.
The cells are cultured in a cell culture medium containing% fetal calf serum (FCS). During this period, the hybridoma cells that have proliferated produce antibodies in the culture medium supernatant, and thus the target antibody can be isolated by ELISA (oxygen immunoassay), RIA (radioisotope immunoassay), plaque assay, agglutination, etc. The presence or absence can be checked, but it is preferable to use the ELISA method. This ELISA method can be performed as follows.

The above A. ELISA for immunizing antigen prepared by
Immobilize in each well of plate A, and then biopolymer proteins such as bovine serum albumin (BSA), ovalbumin (OVA), moss hemocyanin (KLH), and immunoglobulin as blocking agents are immobilized in each well. . This is to prevent the antibody produced by the hybridoma cells from nonspecifically adsorbing to the well during the next operation. After standing for a certain period of time, the supernatant is discarded and each well is washed with a washing solution (which may contain a mixed solution of a phosphate buffer solution and a physiological saline solution and a surfactant). To this, the hybridoma cell culture supernatant is added and left standing for a certain period of time. Similarly, each well is washed with a washing solution, and then an enzyme-labeled antibody, for example, when a mouse is used, an anti-mouse IgG antibody to which an enzyme such as alkaline phosphatase, horseradish peroxidase, or β-galactosidase is bound is added to each well. And leave it for a certain period of time. Wash each well with wash solution,
Next, a substrate solution corresponding to each enzyme used is added and reacted. When the desired antibody is present in the culture supernatant, the color change of the substrate caused by the enzymatic reaction can be confirmed visually or by a plate reader. In this way, hybridoma cells producing the antibody can be obtained.

D. Cloning of hybridoma cells Since it is possible that a mixture of hybridoma cells that are genetically different is present in the well confirmed in step 1, it is necessary to obtain a hybridoma cell group consisting of a single gene by cloning operation. This method includes the limiting dilution method, the single cell manipulation method, the method of picking up colonies on soft agar one by one, the FACS method (Fl
uorecent Activated Cell S
However, it is preferable to use the limiting dilution method because no special equipment is used. This limiting dilution method can be performed as follows.

200 cells / m of the above hybridoma cells
l, 50 / ml, 10 / ml to 0-20
Cell culture medium containing% FCS (RPMI1640, M
EM, DMEM, etc.), and 0.1 ml of each adjusted solution is dispensed into 3, 45, and 48 wells on a 96-well plate. The plate is cultured in a CO ₂ incubator, and wells in which one colony is confirmed to be derived from one hybridoma cell are selected. Whether the monoclonal antibody present in the supernatant of these wells recognizes MA is determined by the method described in C. Re-examine by the ELISA method of. In this way, a hybridoma cell group consisting of a single gene was obtained, and the antibody produced from this cell can be said to be a monoclonal antibody.

E. Preparation of Monoclonal Antibody The preparation of the monoclonal antibody against MA is described in D.
It can be carried out by culturing the hybridoma cells obtained in 1. in a flask or in the abdominal cavity. The culture of the hybridoma cells in the flask was performed with 0-20% FC.
Cell culture medium containing S (RPMI1640, MEM,
DMEM etc.). At this time, the hybridoma cells are allowed to grow to the maximum extent, and then centrifuged to obtain the secreted monoclonal antibody in the supernatant.

The culture of hybridoma cells in the abdominal cavity is
1-10 × 10 ⁶ cells are injected intraperitoneally into an animal that has been administered a mineral oil such as pristane. As the animal species used at this time, it is desirable to use an animal of the same species and strain as the origin myeloma cell so that the myeloma cell used for producing the hybridoma cell can easily proliferate. For example, when a mouse is used, hybridoma cell proliferation is observed in the abdominal cavity after 1 to 2 weeks by this operation, 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.

[0029]

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 Preparation of Antigen for Immunization (1) Preparation of Aminobutylated Methamphetamine In order to bind methamphetamine (MA) to an appropriate protein, for example, the method of Cheng et al. [FEBS L
ETTERS 36 , 339 (1973)], Iwas
aki et al. [Nichiho 41 (3), 217-22]
3, 1987], an amino group was introduced into MA. First, 0.5 g of methamphetamine (MA) hydrochloride powder (trade name: Hiropon) was dissolved in 10 ml of distilled water, and 2N NaOH was added dropwise to bring the pH to about 10. To this, an equal amount of chloroform was added and extracted three times. The obtained chloroform layers were combined, dehydrated, and concentrated. By this operation, the hydrochloric acid group was released, and 0.35 g of methamphetamine was obtained.

The total amount of this methamphetamine was dissolved in 10 ml of dehydrated benzene, 0.86 g of N- (4-bromobutyl) phthalimide and 0.4 g of sodium carbonate were added, and the mixture was added in the presence of nitrogen gas at 80 ° C. for 40 hours. Refluxed. The sodium carbonate was then removed by filtration, an equal volume of 1M HCl was added and further extracted 3 times with an equal volume of benzene. An equal amount of chloroform was added to the aqueous layer and the mixture was extracted 3 times.
The obtained chloroform layers were combined, dehydrated, and concentrated. By this operation, methamphetamine-N-butylphthalimide was obtained. Add 10 ml of ethanol and 0.1
90 ml of hydrazine hydrate (90%) was added, and the mixture was reacted at 80 ° C. for 2 hours in the presence of nitrogen gas. After completion of the reaction, ethanol was distilled off and 1N hydrochloric acid (20 ml) was added. This aqueous solution was extracted 3 times with an equal volume of chloroform, and the aqueous layer was washed with 2
The pH was adjusted to 10 by dropwise addition of N sodium hydroxide.
This aqueous solution was extracted three times with an equal amount of chloroform,
The chloroform layers were combined, dehydrated, and concentrated. In this way, 74 mg of N- (4-aminobutyl) methamphetamine (AB-MA) was obtained. The structure of this AB-MA was confirmed using a mass spectrum and a nuclear magnetic resonance analyzer.

The above-mentioned aminobutylated methamphetamine was used as an immunizing antigen. 10 mg N- (4-aminobutyl) methamphetamine in 1 ml PBS (pH
10 mg BSA after dissolution in 7.2 phosphate buffer)
(Bovine serum albumin) was added, 19.2 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (hereinafter abbreviated as EDC) was further added, and the mixture was stirred at 4 ° C. for 16 hours. All reaction solutions were dialyzed against PBS to remove unreacted EDC and N- (4-aminobutyl) methamphetamine, and N- (4-aminobutyl) methamphetamine-
BSA (abbreviated as MA-AB-BSA-EDC) was obtained. Further, in the same manner, MA-AB-KLH-EDC was obtained by using KLH (Kasai hemocyanin) instead of BSA. In addition, 0.1% glutaraldehyde (GA) was used instead of EDC to bind N- (4-aminobutyl) methamphetamine and BSA or KLH, and M
A-AB-BSA-GA and MA-AB-KLH-GA were obtained, respectively.

Example 2 Preparation of Hybridoma Strain Producing Monoclonal Antibody Against MA (1) Administration of Antigen to Mouse and Preparation of Spleen Cells Administration of the complex of methamphetamine and supporting protein prepared in Example 1 to mouse did. 100 mg MA-A
B-KLH-EDC was dissolved in 0.1 ml of PBS,
The mixture was thoroughly mixed with 0.1 ml of Freund's complete adjuvant, and this emulsion mixture was subcutaneously administered to BALB / c system, 8 week-old mice, 0.2 ml per mouse. This operation was performed on 6 mice once every two weeks for 3 to 6 months. The spleen was aseptically removed from each mouse after administration and washed in RPMI1640 tissue culture medium to remove excess adipose tissue. Next, the new RPMI 1640
After transferring to a medium and shredding with scissors, lymphocytes in the spleen were extruded. The lymphocytes floating in the medium were collected by centrifugation (1,000 rpm, 5 minutes, room temperature), and RPMI1 was collected.
The cells were resuspended in 640 medium and used for the next cell fusion. By this operation, about 1 × 10 ⁸ lymphocytes were obtained from one mouse.

(2) Cell fusion About 4 × 10 ⁷ myeloma cells (6
53 strains) and about 1 × 10 ⁸ lymphocytes obtained in (1) were mixed in RPMI1640 medium and centrifuged (100
The supernatant was removed (0 rpm, 5 minutes, room temperature). 1 ml of 50% polyethylene glycol-RPMI1640 medium was added to the settled myeloma cells and lymphocytes by using a 1 ml pipette for 30 seconds, and the mixture was vigorously shaken to mix. Next, add 1 ml of RPMI1640 medium to 1
Shake vigorously while adding over 8 minutes
RPMI1640 medium was added over 3 minutes, and the supernatant was removed after centrifugation. 30 ml of HAT selection medium (1 x
10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine, RPMI1640 medium containing 20% fetal calf serum) was added, and the cells were well suspended, and then 96 wells were added. It was dispensed to three culture plates. That is, 4.7 × 10 ⁵ cells were dispensed per well, and CO ₂ incubator (5% CO ₂ , 95% air, 3%
The culture was performed at 7 ° C. and a humidity of 100%).

(3) Selection of hybridoma It is confirmed with naked eyes whether or not cells are growing in each well, and whether or not an antibody that recognizes methamphetamine is present in the supernatant of this well is checked. E shown below
It investigated using the LISA method. 96-well ELISA
0.1 mg of MA-AB-BSA-GA prepared in Example 1 was added to a plate (made of polystyrene) for 10 ml of P.
Dissolve in BS and dispense 0.1 ml to each well.
Let stand overnight at 0 ° C. That is, 1 μg of MA-AB-BSA-GA was present per well,
By this operation, MA-AB-BSA-G is attached to the inner wall of the well.
A is adsorbed.

Next, each well of this plate is washed with P for washing.
BS (pH 7.2, containing 0.05% Tween 20)
MA-AB-BSA-G which has not been adsorbed
A was removed. In addition, PBS containing 1% BSA in each well
Was dispensed by 0.1 ml and left at 37 ° C. for 1 hour. Each well was washed 4 times with PBS for washing, and 0.1 ml of the supernatant of the well in which cell growth was confirmed was added. 37 ° C,
After standing for 2 hours, each well was washed 4 times with washing PBS, and an antibody solution against mouse immunoglobulin (alkaline phosphatase binding, diluted to 1/500 in PBS containing 0.5% BSA) was added. 0.1 ml was added and the mixture was allowed to stand at 37 ° C. for 1 hour. PBS for washing each well
Washed 4 times with P-nitrophenyl sodium phosphate.
Substrate solution with 6H ₂ O adjusted to 1 mg / ml 0.1m
After adding 1 l each, the mixture was allowed to stand at room temperature for 30 minutes. In the positive well, sodium p-nitrophenylphosphate is decomposed by the enzymatic activity of alkaline phosphatase, and a yellow color is developed. The reaction was stopped by adding 50 μl of 3M NaOH to each well, and the absorbance at 405 nm was measured using a microplate absorbance meter. By this ELISA method, 51 positive wells producing anti-MA antibody were obtained.

(4) Cloning of hybridoma The hybridoma cells in the 51 positive wells obtained in (3) were monocloned using the limiting dilution method. That is, hybridoma cells were diluted to 200 cells / ml, 50 cells / ml, and 10 cells / ml with RPMI1640 medium containing 20% FCS, and each diluted solution was added to 3, 45, 48 wells on a 96-well plate. 0.1 ml each was dispensed. The plate was cultured in a CO ₂ incubator, and wells in which one colony was confirmed to be derived from one hybridoma cell were selected. Whether the antibody (monoclonal antibody) present in the supernatant of these wells recognizes MA is determined by the above-mentioned ELIS.
It was reexamined by Method A. By this operation, hybridoma cells producing a monoclonal antibody against MA
24 pieces were obtained.

Example 3-Anti-M against MA analogues
Selectivity of A Monoclonal Antibody Regarding the reactivity of the anti-MA monoclonal antibody produced by 24 kinds of hybridoma cells to the structurally similar compound of MA, the ELISA used in the step (3) of Example 2 was used. I examined by law. That is, when a monoclonal antibody produced by each hybridoma is added, a similar compound diluted in multiple stages is added, and to what extent the similar compound reacts with the antigen-antibody reaction between MA bound to the inner wall of the well and the monoclonal antibody, It was examined whether competitive inhibition reaction occurs. The 8C1 strain was selected as a hybridoma cell producing a monoclonal antibody that most specifically reacts with methamphetamine among the 24 types of hybridoma cells. The doubling time of this 8C1 strain was about 24 hours. This hybridoma cell 8
Anti-MA produced by C1 strain (Microtech Lab. No. 13148)
The results of the monoclonal antibody are shown in Table 1.
In this table, the free MA in the sample first shows the binding of MA bound to the inner wall of the well to the monoclonal antibody.
The concentration at which 0% inhibition was obtained was determined, and this value was defined as 100%. Then, the concentrations at which other similar compounds also inhibited this binding by 50% were determined, and the respective ratios with those for MA were shown.

[Table 1] 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.

Example 4-Cultivation of hybridoma cells in mouse abdominal cavity and purification of monoclonal antibody Mice treated with pristane one week before (BALB / c
1 × 10 ^{7 of} the hybridoma cells obtained in Example 3 were intraperitoneally injected into the system (♀, 6 weeks old). After 1-2 weeks, hyperplasia of the abdomen of the mouse was confirmed along with proliferation of hybridoma cells, and ascites was collected from the abdominal cavity using a syringe. By performing this operation every other day for about 1-2 weeks, 10-20 ml of ascites was obtained from one mouse. Add 40 mL of saturated ammonium sulfate solution to this ascites.
%, And the protein fraction containing IgG was precipitated. The precipitate was centrifuged (12,000 rpm,
The cells were collected at 4 ° C. for 20 minutes) and resuspended in 20 ml of PBS. The suspension was dialyzed against PBS overnight to remove ammonium sulfate. In this state, 7-8 mg of the monoclonal antibody can be obtained, which can be sufficiently used for the determination and quantification of MA, but it can be further purified by using DEAE-Sepharose anion exchange resin, G-150 Sephadex gel filtration resin, or the like. .

Example 5-Measurement method of MA using urease labeled monoclonal antibody (1) Binding of enzyme urease to monoclonal antibody Method of Avrameas et al. (Scand. J. Immu)
nol. 8 Suppl. 7.7.1978),
Using glutaraldehyde, urease and the monoclonal antibody were bound by the following method. 5 mg of the monoclonal antibody obtained in Example 4 and urease 2.5
mg (enzyme activity 600-1,200 U / mg) 6 ml
Was suspended in PBS, and glutaraldehyde was added to 0.05%. The mixture was allowed to stand at room temperature for 3 hours to give a urease-labeled anti-MA monoclonal antibody.

(2) Method for measuring MA using ELISA method MA-AB-BSA-GA prepared in Example 1 (1) was adjusted to 0.4 μg per well in 50 mM carbonate buffer (pH 9.6). And adjusted to a 96-well ELISA plate by 0.1 ml each. After standing overnight at 4 ° C, PBS for washing (pH 7.2, 0.05% Tween)
20), 3 times, and then 0.2 ml of PBS (pH 7.2) containing 1% BSA and 0.05% sodium azide was added to each well to prevent nonspecific adsorption of proteins.
We dispensed each. PB for cleaning after standing at 37 ℃ for 2 hours
Wash with S three times. 50 μl prepared in Example 5 (1)
Urease-labeled monoclonal antibody (concentration 2 μg / m
l) and 50 μl of PBS (0.5% BSA, containing 0.05% sodium azide) adjusted to various concentrations of MA as a standard sample were mixed, and a total of 0.1 ml of reaction solution was stored at 37 ° C. for 8 hours. Let stand for a minute. Next, the plate was washed three times with PBS for washing, and 0.1 ml of a substrate solution (bromocresol purple, urea, EDTA, pH 5.0) was added. what if,
If there is no MA in the sample, most of the urease-labeled monoclonal antibodies are M bound to the inner wall of the well.
Since it reacts with A and remains in the well, urea is decomposed and ammonia is produced. Therefore, the pH in the substrate solution is increased, and as a result, bromocresol purple changes from yellow to purple.

On the other hand, when MA is present in the sample, it reacts competitively with the antibody, so that the amount of urease-labeled monoclonal antibody remaining in the well is reduced. for that reason,
No color change of the substrate solution can be confirmed. That is, in the present experimental system using bromocresol purple, the yellow substrate solution turned purple when it was negative, and remained yellow when it was positive. The above color development reaction is completed within 2 minutes, and the enzymatic reaction can be stopped by adding 10 μl of 1% thimerosal solution. The color change can be visually observed or an absorbance measuring instrument for a microplate can be used. The absorbance at 570 nm was measured. By the above operation, all steps could be performed in 11 to 13 minutes, and as the measurement sensitivity, MA up to a concentration of about 2 μg / ml could be determined by visual observation (discoloration by pH indicator).

[Simple kit for measurement and its use] (1) When using a container as a carrier When using an ordinary container (see FIGS. 1 and 2) The MA-supporting protein (this supporting protein is used on the inner wall of the container) For the same as that of MA supporting protein) and a composition comprising the supporting protein and the complex. Preferably, it is applied to a polystyrene test tube. on the other hand,
The urease-labeled monoclonal antibody of the present invention is prepared. The simple assay kit includes at least a test container in which the MA-support complex is applied to the inner wall of the container, a urease-labeled monoclonal antibody solution-containing container, and a substrate solution-containing container containing urea and a pH indicator.

Preferred simple kits for measurement include: 1, a test tube whose inner wall is coated with a MA-supporting protein complex-containing layer (1 to 5 ml, particularly 2 to 3 ml) 2, the urease-labeled monoclonal antibody solution of the present invention Containing container 3, Substrate solution containing container 4, Washing liquid containing washing bottle 5, Quantitative dropper (0.1 to 0.5 ml, especially 0.1.
2 to 0.3 ml) 6. A urine collecting cup or a saliva collecting container.

Urine or saliva (which may be diluted to an appropriate concentration) collected from a suspected stimulant-using agent and a urease-labeled monoclonal antibody solution are put in the above-mentioned test container, and 7 to 10
Incubate for minutes (see Figure 1). After completion of the reaction, the inner solution is discarded and the well is thoroughly washed to remove unreacted urease-labeled monoclonal antibody. Then, a solution for ammonia detection, for example, a pH indicator and a urea-containing solution are added and reacted. In the case of using bromocresol purple as a pH indicator, after about 1 minute, in the case of positive (containing MA), the initial yellow color does not change, and in the case of negative color, it changes to purple (see FIG. 2). When it is desired to fix the color development, for example, a 1% thimerosal solution can be added.

In the case of using a container capable of sucking as a carrier (see FIGS. 3 and 4) MA-supporting protein on the inner wall of the container capable of sucking as shown in FIGS. The same as described above) is applied and a composition comprising a supporting protein is applied. Preferably, it is applied to the inner wall of the polystyrene pipette container. on the other hand,
An enzyme-labeled monoclonal antibody solution of the present invention (for example, urease-labeled monoclonal antibody) is prepared. The simple assay kit includes at least a container for pipette having the inner wall coated with the MA-supporting protein complex and a container containing an enzyme-labeled monoclonal antibody solution.

Preferred simple kits for measurement are: 1, a pipette type container whose inner wall is coated with a MA-supporting protein complex-containing layer 2, a container containing the enzyme-labeled monoclonal antibody solution of the present invention 3, a substrate solution containing container 4 , A washing liquid-containing washing bottle 5, a quantitative dropper 6, a urine collecting cup or a saliva collecting container.

Urine or saliva (which may be diluted to an appropriate concentration) collected from a suspected stimulant use agent and the enzyme-labeled monoclonal antibody solution are aspirated and injected by connecting the pipette container to a syringe or the like, and then 7 to 10 Incubate for minutes (see Figure 3). After the reaction is completed, the inner solution is discarded and the well is thoroughly washed to remove the unreacted enzyme-labeled monoclonal antibody. Then, a solution for ammonia detection, for example, a pH indicator and a urea-containing solution are added and reacted. In the case of using bromocresol purple as a pH indicator, after about 1 minute, in the case of positive (containing MA), the initial yellow color does not change, and in the case of negative color, it changes to purple (see FIG. 4). When it is desired to fix the color development, for example, a 1% thimerosal solution can be added.

(2) When a stick is used as a carrier (see FIGS. 5 to 6) A stick such as a polystyrene stick and MA-
A composition comprising the support protein complex and the support protein is applied to form a stick for measurement. On the other hand, the urease-labeled monoclonal antibody of the present invention is prepared. The simple assay kit comprises at least a measuring stick in which the MA-support complex is applied to the inner wall of the container, a container containing the urease-labeled monoclonal antibody solution of the present invention, and urea and p.
Includes a substrate solution containing container containing an H indicator. As a preferable simple kit for measurement, 1, a stick having a MA-supporting protein complex-containing layer 2, a container containing the enzyme-labeled monoclonal antibody of the present invention 3, a substrate solution containing container 4, a washing test tube 5, a quantification dropper 6 , A urine collection cup or a saliva collection container.

Urine or saliva (which may be diluted to an appropriate concentration) collected from a suspected person using a stimulant and an enzyme-labeled monoclonal antibody solution are placed in an arbitrary test container, for example, a test tube, and the stick is immersed therein. . Immersion time is 7-10
It reacts sufficiently in a minute (see FIGS. 5 to 6). After completion of the reaction, the stick is immersed in a test tube containing a cleaning solution for cleaning. This washing method is repeated 1 to 4 times. Then, separately,
The substrate solution is placed in a test tube, and the washed stick is immersed therein. After about 1 minute of the immersion treatment, the solution in the test tube remains yellow when positive and turns purple when negative (see FIG. 6). When it is desired to fix the color development, for example, a 1% thimerosal solution can be added.

[0051]

[Effect] The present invention provides a method for detecting methamphetamine by a simple color reaction of discoloration of a pH indicator by skillfully utilizing the reaction that ammonia is generated in the system by the reaction of urease-labeled monoclonal antibody and urea. To do. Since it can be detected by using a pH indicator, the detection kit is of a portable size and can be always installed in a police car, etc., and its usage is simple, so no special training is required. It can be used correctly by all police officers and can immediately inspect and arrest stimulant users.

[Brief description of drawings]

FIG. 1 is a drawing showing one use mode of the MA measurement simple kit of the present invention, and shows the entire use procedure thereof with reference to FIGS. 1 and 2. FIG.

FIG. 2 shows a continuation of the use procedure of FIG.

FIG. 3 is a drawing showing another mode of use of the MA measurement simple kit of the present invention, and shows the entire procedure of use with reference to FIGS. 3 and 4.

FIG. 4 shows a continuation of the use procedure of FIG.

FIG. 5 is a drawing showing another usage mode of the MA measurement simple kit of the present invention, and shows the entire usage procedure thereof with reference to FIGS. 5 and 6.

FIG. 6 shows a continuation of the use procedure of FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 G 8310-2J 33/543 8310-2J 33/577 B 8310-2J // (C12P 21/08 C12R 1:91) (72) Inventor Toyoji Hozumi 3-5 Kasumigaseki, Chiyoda-ku, Tokyo Within Showa Shell Sekiyu Co., Ltd. (72) Sumiko Hara 3-5-2 Kasumigaseki, Chiyoda-ku, Tokyo No. Showa Oil Co., Ltd. (72) Inventor Takao Matsumoto 3-5 Kasumigaseki, Chiyoda-ku, Tokyo Showa Oil Oil Co., Ltd.

Claims (11)

[Claims] 1. A hybridoma strain of Microorganism Research Institute No. 13148. 2. A methamphetamine immunizing antigen is inoculated into a mammal to obtain lymphocytes capable of producing anti-methamphetamine antibody, the lymphocytes are fused with myeloma cells, and the obtained fused cells are cultured, The method for producing a hybridoma strain according to claim 1, wherein a monoclonal antibody which shows a specific immune reaction to methamphetamine is produced. 3. A monoclonal antibody produced by the hybridoma strain according to claim 1, which shows a specific immune reaction to methamphetamine. 4. The method for producing a monoclonal antibody according to claim 3, which comprises culturing the hybridoma strain according to claim 1. 5. A carrier obtained by (a) supporting a complex of methamphetamine and a supporting protein, and (b) labeling a monoclonal antibody showing a specific immunoreaction with methamphetamine with urease. The urease-labeled monoclonal antibody and (c) methamphetamine in the measurement sample are competitively reacted, and after the reaction is completed, (d) urea is reacted with the urease-labeled monoclonal antibody bound to the carrier of (a) to produce A method for detecting methamphetamine, which comprises detecting an ammonia component. 6. The method for detecting methamphetamine according to claim 5, wherein the generated ammonia component is detected by a pH indicator. 7. The method for detecting methamphetamine according to claim 5, wherein the measurement sample is urine or saliva of a subject. 8. (i) A container containing a urease-labeled monoclonal antibody, which is obtained by labeling a monoclonal antibody that shows a specific immune reaction to methamphetamine with urease, (ii) a complex of methamphetamine and a supporting protein A carrier carrying (iii) a substrate solution-containing container containing urea and a pH indicator, or a substrate solution-containing container containing urea and a pH indicator-containing container,
A portable methamphetamine detection kit containing the. 9. The portable methamphetamine detection kit according to claim 8, which contains a quantitative dropper and / or a specimen collection container. 10. The portable methamphetamine detection kit according to claim 8 or 9, wherein the (ii) is a container in which a complex of methamphetamine and a supporting protein is fixed to the inner wall thereof. 11. The portable methamphetamine detection kit according to claim 8 or 9, wherein the (ii) is a stick having a complex of methamphetamine and a supporting protein immobilized on its surface layer.