JPH057497A - New monoclonal antibody, new hybridoma producing the same and production thereof - Google Patents

Abstract

PURPOSE:To provide the subject antibody exhibiting specific immune reactions against both methanephetamine and N,N'-dibenzylethylenediamine, and capable of simply measuring the compounds in high sensitivity and selectivity without employing the methanephetamine which is a stimulant drug. CONSTITUTION:Methanephetamine is reacted with ethyl bromoacetate, etc., in the presence of sodium carbonate in ethanol. The produced N- carboxylmethylated methanephetamine of the formula is bonded to bovine serum albumin in the presence of a carbodiimide compound to prepare an antibody for immunizing against the methanephetamine. The antibody is inoculated in a mammalian to immunize the mammalian. Lymphocytes having an ability to produce methanephetamine-resistant antibodies are separated from the spleen of the mammalian, and subsequently fused with myeloma cells to form hybridomas. The hybridomas are cultured in a HAT medium and subjected to a cloning treatment. The monoclonal antibody is cultured to provide the objective monoclonal antibody exhibiting specific immune reactions against both the methanephetamine and N,N'-dibenzylethylenediamine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody which specifically recognizes both methamphetamine (hereinafter sometimes referred to as MA) and N, N′-dibenzylethylenediamine (hereinafter sometimes referred to as DBED). An antibody and a method for producing the same.

[0002]

2. Description of the Related Art As the number of methamphetamine-related cases has increased, the need for methamphetamine users to be easily, quickly and accurately recognized has increased, and a simple, highly sensitive and highly selective method for measuring methamphetamine has been desired. There is. JP-A-1-96
In 198, it is proposed to obtain a monoclonal antibody having a very high specific reactivity to methamphetamine (MA) which is a stimulant by culturing a cell line obtained by immunizing an animal. ing. And
At present, as a measuring system using a monoclonal antibody that specifically recognizes only MA, a competitive assay method based on the ELISA method has been commercialized. However, in this measurement system method, it is necessary to use MA itself in a product, and in reality, these products are not allowed to be manufactured, sold, or used according to the Japanese stimulant agent control method.

[0003]

[Objective] The object of the present invention is to produce a monoclonal antibody that selectively and specifically recognizes a specific substance that does not conflict with Japanese law when inducing a monoclonal antibody against MA, and the measurement kit contains this antibody and the specific substance. Is to provide a monoclonal antibody for detecting MA and a method for producing the same, which solves the above problems.

[0004]

[Structure] The first of the present invention is methamphetamine and N,
The present invention relates to a monoclonal antibody characterized by exhibiting a specific immune reaction to both N'-dibenzylethylenediamine. A second aspect of the present invention relates to a hybridoma strain having the ability to produce the above-mentioned monoclonal antibody. A third aspect of the present invention relates to a method for producing a monoclonal antibody, which comprises culturing the hybridoma strain. A fourth aspect of the present invention is to inoculate a mammal with an immunizing antigen of methamphetamine to obtain lymphocytes having an anti-methamphetamine antibody-producing ability, to fuse the lymphocytes with myeloma cells, and to culture the obtained fused cells. Then
The hybridoma strain according to claim 2, which has the ability to produce a monoclonal antibody that shows a specific immune reaction to both methamphetamine and N, N'-dibenzylethylenediamine, is selected from the above. It relates to a method for producing the hybridoma strain described.

[Manufacturing Process of Monoclonal Antibody] A. Preparation of immunizing antigen Methamphetamine (MA) is aminobutylated or carboxymethylated to form aminobutylated or carboxymethylated methamphetamine, and an arbitrary supporting protein is bound to the complex to obtain a complex, which is used as an immunizing antigen. did. Examples of the supporting protein include bovine serum albumin, ovalbumin, Jinkasai 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, etc. are used. To be B. Preparation of lymphocytes The immunizing antigen is administered to a mammal (eg, mouse, rat, etc.) every 3 to 5 times every other week, and after confirming that antibodies against the antigen are sufficiently produced, the blood lymph of the animal is confirmed. Lymphocytes are obtained from nodes, spleen, etc. At this time, it is preferable to administer an adjuvant (including Mewban, killed tuberculosis, nucleic acid, etc.) as an immunostimulant together with the antigenic substance to the animal as an emulsion. As a means for confirming the production of antibodies, venous blood is collected from the immunized animal, and II.
Means for determination can be mentioned by using the ELISA method in the section of hybridoma selection in (3). C. Examples of myeloma cells used for cell fusion and selection of hybridoma strain include 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 and the myeloma cells of the immunized animal as described above are mixed at about 2 to 10: 1, and the mixture is centrifuged to obtain a mixed precipitate of lymphocytes and myeloma cells. A cell culture medium (R containing polyethylene glycol (PEG) or Sendai virus (HVJ)
PMI1640, MEM, DMEM, etc.) can be added and suspended.
It is preferable to use 0% to 60% PEG (molecular weight 1000 to 8000). Hybridoma strains were selected by centrifuging the cell suspension after fusion to remove the supernatant, and adding hypoxanthine, aminopterin, thymidine (HAT)
It can be carried out by resuspending in a cell culture medium containing 15% to 20% 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, aminopterin (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 proliferated produce antibodies in the medium supernatant, and
The presence or absence of the desired antibody can be examined by using (enzyme immunoassay) method, RIA (radioisotope immunoassay) method, plaque assay method, agglutination method and the like, but it is preferable to use ELISA method. This ELISA method can be performed as follows. A. The immunizing antigen prepared in 1. was immobilized in each well of an ELISA plate, and then bovine serum albumin (BSA), ovalbumin (OVA), and Jinkasai hemocyanin (KL) were used as blocking agents.
H), a biopolymer protein such as immunoglobulin is 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 to stand for a certain period of time. Similarly, each well was washed with a washing solution, and then an enzyme-labeled antibody, for example, anti-mouse IgG when a mouse was used.
An antibody to which an enzyme such as alkaline phosphatase, horseradish peroxidase, or β-galactosidase is bound is added to each well and allowed to stand for a certain period of time. Each well is washed with a washing solution, and then a substrate solution corresponding to the 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 C. 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 limiting dilution method, single cell manipulation method, picking colonies on soft agar one by one, FACS method (Fluorecent
Activated Cell Sorter), but it is preferable to use the limiting dilution method because no special equipment is used.
This limiting dilution method can be performed as follows. Cell culture medium (RPMI1640, MEM, DM) containing 0 to 20% FCS at 200 cells / ml, 50 cells / ml, and 10 cells / ml of the above hybridoma cells.
EM, etc.) and dispense 0.1 ml of each adjustment solution 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 antibodies present in the supernatant of these wells recognize MA was determined by C.I. 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 Antibodies For the preparation of monoclonal antibodies against MA, see 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 is performed by a cell culture medium (RPMI1640, MEM, DM) containing 0 to 20% FCS.
EM 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. For culturing the hybridoma cells in the abdominal cavity, 1 to 10 × 10 ⁶ cells are injected into the abdominal cavity of an animal to which mineral oil such as pristane is administered. 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 is present in the ascites, the ascites is collected from the abdominal cavity, and salting out, dialysis,
The monoclonal antibody can be separated and purified by an operation such as ion exchange chromatography or affinity chromatography.

[0006]

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 I.D. Preparation of immunizing antigen 1. Preparation of aminobutylated methamphetamine To bind methamphetamine (MA) to an appropriate protein, for example, the method of Cheng et al. [FEBS LETTERS 36,3
39 (1973)], an amino group was introduced into MA.
400 mg of methamphetamine (MA) was dissolved in 40 ml of dehydrated ethanol and 2.272 g of N- (4-
Bromobutyl) phthalimide and 852 mg of sodium carbonate were added, and the mixture was refluxed for 4 days. Then 0.5 ml of 85
% Hydrous hydrazine was added and stirred for 2 days. After adding 1 N hydrochloric acid to the reaction solution to make it acidic, the generated precipitate was removed by filtration. The filtrate was extracted 3 times with 50 ml of chloroform, and the aqueous layer was adjusted to pH 10 with 1N sodium hydroxide, and then extracted 4 times with 50 ml of chloroform. The chloroform layers were combined, dehydrated with magnesium sulfate, and chloroform was distilled off under reduced pressure. In this way, 1.27 g of N- (4-aminobutyl) methamphetamine was obtained.

[Chemical 1] 2. Preparation of carboxymethylated methamphetamine In order to bind MA to an appropriate protein, for example, In
A carboxyl group was introduced into MA according to the method of Ayama et al. [Chem. Pharm. Bull 28, 2779 (1980)]. The following reactions were all performed at room temperature. 600 mg of methamphetamine, 852 mg of sodium carbonate and 1.5 ml of ethyl bromoacetic acid were dissolved in 40 ml of ethanol and refluxed under nitrogen gas for 5 days. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. Next, 30 ml of 5% potassium hydroxide-methanol solution was added, and the mixture was stirred for 2 days under nitrogen gas filling. After the reaction is complete, add 2
It was acidified with N hydrochloric acid and concentrated under reduced pressure to about 1 ml. To this, add 5 ml of saturated aqueous sodium chloride solution,
It was extracted 5 times with ml of chloroform. The chloroform layer was dried over magnesium sulfate and concentrated under reduced pressure. In this way, 691.1 mg of N-carboxymethylmethamphetamine was obtained.

[Chemical 2] 3. Immunization antigen or enzyme immunoassay (hereinafter referred to as ELIS
The preparation of the antigen used for A) was carried out using the above aminobutylated or carboxymethylated methamphetamine. 25 mg of N- (4-aminobutyl) methamphetamine was dissolved in 1 ml of PBS (10 mM-phosphate buffer solution, 150 mM-sodium chloride, pH 7.2), and the pH was adjusted to 6.5 to 7. Adjusted to 0. On the other hand, 50 mg of BSA was dissolved in 1 ml of PBS and then added dropwise to the above solution while stirring. 2 to 5
μl of 25% glutaraldebit PBS solution was added, and the mixture was stirred at room temperature for 90 minutes. Next, the whole solution is kept at 4 ° C for 24 hours.
Dialyzed against PBS for hours. This gave 70 mg of N- (4-aminobutyl) methamphetamine-bovine serum albumin-glutaraldehyde complex (MA-AB
-BSA-GA) was obtained. Or 25 mg KL
H was dissolved in 1 ml of distilled water, 15 mg of N- (4-aminobutyl) methamphetamine dissolved in 0.5 ml of dimethylformamide was added dropwise, and the mixture was stirred. 1m to this
500 mg 1-ethyl-3-dissolved in 1 liter distilled water
(3-Dimethylaminopropyl) carbodiimide (ED
C) was added and stirred at room temperature for 6 hours. 4 for all reaction solutions
2 days at ℃, dialyzed against distilled water to remove EDC, 3
4 mg of N- (4-aminobutyl) methamphetamine-
Jinkasai hemocyanin (MA-AB-KLH-EDC) was obtained. Further, in the same operation, MA-AB-OVA was obtained by using ovalbumin (OVA) or BSA instead of KLH.
-EDC and MA-AB-BSA-EDC were obtained, respectively. Furthermore, instead of N- (4-aminobutyl) methamphetamine, N-carboxymethylmethamphetamine and B
Using SA, KLH, MA-CM-BSA-EDC,
MA-CM-KLH-EDC was obtained respectively. II. Preparation of Hybridoma Strain Producing Monoclonal Antibody Against MA (1) Administration of Antigen to Mice and Preparation of Spleen Cells I. The complex of methamphetamine and the supporting protein prepared in 1. was administered to 4 mice. For example, 0.1m
g of MA-CM-KLH-GA was dissolved in 0.1 ml of PBS and mixed well with 0.1 ml of Freund's complete adjuvant, and 0.2 ml of this emulsified mixture was mixed with BALB / c system,
♀, 7-week-old mice were intraperitoneally administered. The spleen was aseptically removed from each mouse after administration and washed in RPMI1640 tissue culture medium to remove excess adipose tissue. Next, the cells were transferred to a new RPMI1640 medium and shredded with scissors, and then the lymphocytes in the spleen were pushed out using a rubber stopper.
Centrifuge floating lymphocytes (1,000 rpm,
The cells were collected for 5 minutes at room temperature), resuspended in RPMI1640 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 (Boehringer Mannhei) was added to the precipitated myeloma cells and lymphocytes.
m company) -RPMI1640 medium was vigorously shaken and mixed using a 1 ml pipette while gradually adding for 30 seconds. Then, 1 ml of RPMI1640 medium was added over 1 minute while shaking vigorously, and further 8 ml of RPM was added.
I1640 medium was added over 3 minutes, and the supernatant was removed after centrifugation. 30 ml of HAT selection medium (1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 ×
RPMI containing 10 ^-5 M thymidine and 20% fetal bovine serum
1640 medium) and the cells were well suspended, and then 96
It dispensed to three culture plates of a well. That is, about 4.7 × 10 ⁵ cells were dispensed per well and cultured using a CO ₂ incubator (5% CO ₂ , 95% air, 37 ° C., humidity 100%). (3) Selection of hybridoma It is confirmed by naked eyes whether cells are proliferating in each well, and it is further shown by the following EL whether or not an antibody that recognizes methamphetamine is present in the supernatant of this well.
It examined using the ISA method. 96-well ELISA plate (polystyrene, ImmunolonDyn
technology), 0.1 mg MA prepared in (I)
-AB-BSA-GA was dissolved in 10 ml of 50 mM carbonate buffer (pH 9.6), 0.1 ml was dispensed into each well, and the mixture was allowed to stand at 4 ° C overnight. That is, 1 μg of MA-AB-BSA-GA is present per well, and this operation causes adsorption to the inner wall of the well. Next, each well of this plate was washed with PBS (pH 7.
2, containing 0.05% Tween 20), the non-adsorbed MA-AB-BSA-GA was removed. Furthermore, 0.1m PBS containing 0.5% BSA is added to each well.
It was dispensed in liters 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. After standing at 37 ° C. for 2 hours, each well was washed 4 times with PBS for washing, and an antibody solution against mouse immunoglobulin (alkaline phosphatase binding, Sigma, PB containing 0.5% BSA was used).
0.1 ml of S diluted to 1/500) was added and left at 37 ° C. for 1 hour. PB for washing each well
The substrate solution washed with S four times and adjusted to 1 mg / ml with sodium P-nitrophenylphosphate · 6H ₂ O was added to 0.1%.
After adding each ml, the mixture was allowed to stand at room temperature for 30 minutes. In the positive wells, 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. Next, the supernatant of the positive well was subjected to a competitive inhibition test using MA (with the supernatant of each well described above, MA was added at 1 μg / ml). 51 positive wells producing antibody were obtained by these ELISA methods. (4) Cloning of hybridoma The hybridoma cells in the 51 positive wells obtained in (3) were monocloned using the limiting dilution method. That is, the hybridoma cells were diluted with RPMI1640 medium containing 20% FCS so as to have 200 cells / ml, 50 cells / ml, and 10 cells / ml, 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. III. Examination of selectivity of anti-MA monoclonal antibody against a compound similar to MA, and 24 hybridoma cells producing the monoclonal antibody of claim 1 (selection of claim 2) showing a specific immune reaction to both MA and DBED. ELI used in the step (3) of (II) for the reactivity of the anti-MA monoclonal antibody produced by Escherichia coli with the structurally similar compound of MA.
It was examined by the SA method. That is, when a monoclonal antibody produced by each hybridoma was added, a similar compound diluted in multiple steps was added, and how much the similar compound competed with the antigen-antibody reaction between MA on the plate and the monoclonal antibody. It was investigated whether or not an inhibition reaction occurs. As a result, among the monoclonal antibodies obtained from 24 kinds of hybridoma strains, hybridoma strains producing a monoclonal antibody represented by 4B2 strain or 2H3 strain which reacts only with MA and DBED were selected. In Table 1, the reactivity with MA is set to 100 and the reactivity with other similar compounds is shown. In the table, MA is methamphetamine DBED, N, N'-dibenzylethylenediamine A, amphetamine MPA, methoxyphenamine NOR-EP (+), norephedrine (+) OH-MA, P-hydroxymethamphetamine OH. -NOREP, P-hydroxynorephedrine OH-EP, P-hydroxyephedrine OH-A, P-hydroxyamphetamine ME, methylephedrine NOR-EP (-), norephedrine (-) EP, ephedrine PHE Phentermine MES is Mescaline.

[Table 1]

[Table 2] IV. Examination of the class and subclass of the selected anti-MA-DEBD monoclonal antibody (Claim 1) To examine which class or subclass the monoclonal antibody produced by the 2H3 strain belongs to among the obtained hybridoma cells, Eight anti-mouse classes, subclasses (Ig
G ₁ , IgG ₂ a, IgG ₂ b, IgG ₃ , IgM, Ig
A, κ type L chain, λ type L chain) antibody, II. It was examined by the ELISA method of (3). As a result, the monoclonal antibody produced by the 2H3 strain is an IgG having a κ L chain.
It was found that the antibody belongs to ₁ . V. Culturing the hybridoma cells according to claim 2 in the abdominal cavity of a mouse and purifying the monoclonal antibody according to claim 1 A mouse (BALB / c) treated with pristane one week before.
System, ♀, 6 weeks old), II. The hybridoma cells obtained in 1. were intraperitoneally injected at 1 × 10 ⁷ cells. 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. A saturated ammonium sulfate solution was added to this ascites fluid so as to be 40% to precipitate a protein fraction containing IgG. The precipitate was centrifuged (12,000 rpm, 4 ° C,
20 minutes) and resuspended in 20 ml 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 and 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. .

[0007]

[Effect] Since the novel monoclonal antibody of the present invention is produced from a novel hybridoma strain and can specifically react not only with methamphetamine but also with N, N'-dibenzylethylenediamine, it is a stimulant. N, N'-dibenzylethylenediamine can be used instead of methamphetamine used in the assay kit for detection. Therefore, a measuring kit using the monoclonal antibody of the present invention contains MA in the kit.
Since it is not used, domestic sales without touching domestic law,
It can be used, and methamphetamine can be easily measured with high sensitivity.

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[Procedure amendment]

[Submission date] July 29, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

[Manufacturing Process of Monoclonal Antibody] A. Preparation of immunizing antigen Methamphetamine (MA) is aminobutylated or carboxymethylated to form aminobutylated or carboxymethylated methamphetamine, and an arbitrary supporting protein is bound to the complex to obtain a complex, which is used as an immunizing antigen. did. Examples of the supporting protein include bovine serum albumin, ovalbumin, Jinkasai 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, etc. are used. To be B. Preparation of lymphocytes The immunizing antigen is administered to a mammal (eg, mouse, rat, etc.) every 3 to 5 times every other week, and after confirming that antibodies against the antigen are sufficiently produced, the blood lymph of the animal is confirmed. Lymphocytes are obtained from nodes, spleen, etc. 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. As a means for confirming the production of antibodies, venous blood is collected from the immunized animal, and II.
Means for determination can be mentioned by using the ELISA method in the section of hybridoma selection in (3). C. Examples of myeloma cells used for cell fusion and selection of hybridoma strain include 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 and the myeloma cells of the immunized animal as described above are mixed at about 2 to 10: 1, and the mixture is centrifuged to obtain a mixed precipitate of lymphocytes and myeloma cells. A cell culture medium (R containing polyethylene glycol (PEG) or Sendai virus (HVJ)
PMI1640, MEM, DMEM, etc.) can be added and suspended.
It is preferable to use 0% to 60% PEG (molecular weight 1000 to 8000). Hybridoma strains were selected by centrifuging the cell suspension after fusion to remove the supernatant, and adding hypoxanthine, aminopterin, thymidine (HAT)
It can be carried out by resuspending in a cell culture medium containing 15% to 20% fetal calf serum (FCS) and dispensing this suspension into a culture plate. The hybridoma cells selected by this operation were further mixed with hypoxanthine, thymidine (HT) and 10% to 20% fetal calf serum (FC).
Culture in a cell culture medium containing S), and finally 10%
Culture in cell culture medium containing ~ 20% fetal calf serum (FCS). During this period, the hybridoma cells that have proliferated produce antibodies in the medium supernatant, and therefore, ELISA (enzyme immunoassay) method, RIA (radioisotope immunoassay) method are used.
Although the presence or absence of the desired antibody can be examined by using the method, plaque assay method, agglutination method, etc., it is preferable to use the ELISA method. This ELISA method can be performed as follows. A. EL for immunization antigen prepared by
Immobilize in each well of ISA plate, and then immobilize biopolymer proteins such as bovine serum albumin (BSA), ovalbumin (OVA), Jinkasai hemocyanin (KLH) and immunoglobulin as blocking agents 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 to stand for a certain period of time. Similarly, each well was washed with a washing solution, and then an enzyme-labeled antibody, for example, anti-mouse IgG antibody when a mouse was used, was added to alkaline phosphatase or horseradish peroxidase,
A product to which an enzyme such as β-galactosidase is bound is added to each well and allowed to stand for a certain period of time. Each well is washed with a washing solution, and then a substrate solution corresponding to the 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 C. 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 limiting dilution method, single cell manipulation method, picking colonies on soft agar one by one, FACS method (Fluorecent
Activated Cell Sorter), but it is preferable to use the limiting dilution method because no special equipment is used.
This limiting dilution method can be performed as follows. Cell culture medium (RPMI1640, MEM, DM) containing 0 to 20% FCS at 200 cells / ml, 50 cells / ml, and 10 cells / ml of the above hybridoma cells.
EM, etc.) and dispense 0.1 ml of each adjustment solution 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 antibodies present in the supernatant of these wells recognize MA was determined by C.I. 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 Antibodies For the preparation of monoclonal antibodies against MA, see 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 is performed by a cell culture medium (RPMI1640, MEM, DM) containing 0 to 20% FCS.
EM 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. For culturing the hybridoma cells in the abdominal cavity, 1 to 10 × 10 ⁶ cells are injected into the abdominal cavity of an animal to which mineral oil such as pristane is administered. 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 is present in the ascites, the ascites is collected from the abdominal cavity, and salting out, dialysis,
The monoclonal antibody can be separated and purified by an operation such as ion exchange chromatography or affinity chromatography.

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Claims (4)

[Claims] 1. A monoclonal antibody which exhibits a specific immune reaction to both methamphetamine and N, N'-dibenzylethylenediamine. 2. A hybridoma strain having the ability to produce the monoclonal antibody according to claim 1. 3. A method for producing a monoclonal antibody, which comprises culturing the hybridoma strain according to claim 2. 4. A methamphetamine immunizing antigen is inoculated into a mammal to obtain a lymphocyte having an ability to produce an anti-methamphetamine antibody, the lymphocyte and the myeloma cell are fused, and the obtained fused cell is cultured. The hybridoma strain according to claim 2, which has the ability to produce a monoclonal antibody that shows a specific immune reaction to both methamphetamine and N, N'-dibenzylethylenediamine, is selected from the above. A method for producing the hybridoma strain described.