JP2605418B2 - Antibodies with high affinity for hapten antigens - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to improving the performance of antibodies that play the most important role in immunological detection methods useful in the medical diagnostic field, chemical analysis, and the like. In particular, the present invention relates to the production of antibodies having high affinity for low-molecular-weight compounds, ie, haptens, which cannot cause an immune response by themselves.

More specifically, the present invention relates to the production of an antibody and a monoclonal antibody having a high affinity for methamphetamine which is a kind of a stimulant having a central nervous system stimulating action.

2. Description of the Related Art When detecting a trace component in blood or a specific component in the atmosphere, it is necessary to reliably detect a specific trace component from a very large number of impurities. For this purpose,
In recent years, immunological detection methods based on antibody reactions have been actively studied.

Immunological detection methods are broadly classified into radioimmunoassay (RIA) using radioisotopes and enzyme immunoassay (EIA) using enzymes. EIA is advantageous in terms of safety and simplicity.

Here, a general enzyme linked immunosorbent assay (ELISA) method as EIA will be described. In FIG. 1, reference numeral 1 denotes a microplate made of plastic polystyrene or the like that nonspecifically adsorbs proteins.
2 is a solid-phase antigen in which an appropriate functional group is introduced into an antigen, which is a non-measurement substance, and chemically bound to a protein via this.
Adsorbed on the microplate 1. Reference numeral 3 denotes an antibody capable of binding to a non-measurement substance, which is equilibrium-bound to the solid-phase antigen 2. That is, the antibody 3 is immobilized on the microplate 1 via the solid-phase antigen 2. In this state, 4
When the antigen as the analyte is introduced, the solid phase antigen 2 and the antigen 4 as the analyte are competitively bound to the antibody 3. Therefore, part of the antibody 3 is the antigen 4
As a result, some antibodies 3 are not immobilized on the microplate 1. Naturally, the more the antibody 4 that is the substance to be measured, the more the antibody 3 not immobilized on the microplate 1. By performing the washing here, the antibodies 3 not immobilized on the microplate 1 can be removed. After removing the antibody 3 not immobilized on the microplate 1, the antibody 5 having a binding ability to the antibody 3 is added. However, antibody 5 is chemically bound to enzyme 6 at a fixed ratio. After removing the antibody 5 not bound to the antibody 3 by washing, the activity of the enzyme 6 is measured. Since there is a positive correlation between the activity and the amount of enzyme 6, the amount of enzyme 6 can be quantified by measuring the activity. If the amount of the enzyme 6 is known, the antibody 3 immobilized on the microplate 1 via the solid-phase antigen 2 retroactively
Can be quantified, and the amount of antigen 4
Can be determined. At this time, the first factor that determines the detection sensitivity is the affinity of the antibody 3 for the antigen 4 which is the substance to be measured.

An antibody is a type of protein produced by an organism and has a property of selectively binding only to a specific substance, that is, an antigen.
Antigens are broadly classified into two types depending on the method of preparing antibodies that bind to the antigen. One is that an antibody can be produced by directly injecting it into an animal body, and a macromolecule having a molecular weight of tens of thousands or more usually corresponds to this. The other has a relatively small molecular weight and can be an immunogen that induces the production of antibodies only after chemically binding to an appropriate protein. In particular, the latter antigens are called haptens. Regarding the method for producing an antibody against an antigen which is a hapten, for example, the following literature (Haptens and Carriers, O. Makera and IJT Separa, Handbook of Experimental Immunology Force Edition, Vol. 1, Chap 3, Edited by DM Veer , Blackwell Scientific Publications, Oxford, 1986.
Carriers, O.Makela and IJTSeppala, Handbook of E
xperimental Immunology 4th edition, Vol.1, Chap 3,
ed by DMWeir, Blackwell Scientific Publification
s, Oxford, 1986.). That is, if a functional group capable of chemical bonding is present in the hapten, the functional group is used to bind to a protein to produce an immunogen. If there is no functional group in the target hapten, a hapten derivative having an appropriate functional group such as an amino group, a carboxyl group, or a hydroxyl group and a spacer having an alkyl chain of about 1 to 10 carbon atoms was synthesized. Thereafter, it is common to obtain an immunogen by binding to an appropriate protein with a crosslinking reagent or the like.
To give a more specific example, Takami et al. Reported on the production of antibodies using methamphetamine (MA), a type of stimulant, as the hapten antigen (Takami, Fukuda and Takahashi, Japanese Journal of Legal Medicine, Takami , Fukuda and Takahashi, Jp
n, J., Legal Med., 37 (4), 417, 1983). That is, MA
Aminobutyl groups are introduced into secondary amines of the above to make aminobutylmethamphetamine (ABMA), which is bound to proteins to form an immunogen.

Problems to be Solved by the Invention However, it has been revealed by the present inventors that antibodies prepared using the above-mentioned immunogen have a high affinity for the antigen derivative ABMA rather than the target antigen MA. Became. That is, the antibody obtained by the conventional method is an antibody against the antigen derivative. The antigen derivative has a different structure from the original target antigen due to the introduction of functional groups, spacers, etc., and the problem that it is difficult to produce antibodies with high affinity for the target antigen is highlighted. Was.

For this reason, when an antibody originally obtained by a conventional method is used to measure an antigen originally intended in the above-mentioned ELISA method as a substance to be measured, for example, even when a large amount of the antigen to be measured is introduced, micro- Since the amount of the antibody not immobilized on the prolate does not increase, it becomes impossible to detect the antigen to be measured with high sensitivity.

Means for Solving the Problems In order to solve the above problems, it was considered that a substance having a high similarity to the target antigen in a state where the functional group and the spacer were introduced was used as a starting material for the synthesis of the antigen derivative.

Specifically, starting from a substance in which the alkyl group in the target antigen molecule has been replaced by a hydrogen atom, a functional group and a spacer capable of binding to a protein are introduced at the position of the hydrogen atom, and the protein is linked via this functional group. It is conceivable to produce an immunogen obtained by binding to

More specifically, methamphetamine (MA), which is a type of stimulant and has a central nervous system stimulating effect, is used as a target antigen, and in order to produce an antibody having a high affinity for such an antigen, the N-alkyl group of MA is used. Aminobutylamphetamine (ABAP), which is an antigen derivative in which an aminobutyl group was introduced as a functional group capable of binding to a protein and a spacer to amphetamine (AP) having a structure in which a certain methyl group was substituted with a hydrogen atom, was used. The chemical structures of AP, ABAP, MA and ABMA are shown below.

It can be seen that MA and ABAP have high similarity in chemical structure.

Other preferred antigen derivatives include the following structural formula (n
Represents an integer). n is an integer, particularly preferably n is an integer of 1 to 10. When n is 4, the following structural formula shows ABAP.

These amphetamine derivatives can be added to AP, for example, N-
It can be easily obtained by reacting N-haloalkylphthalimide such as bromobutylphthalimide to give N- (4-phthalimidylalkyl) amphetamine, and then treating this with hydrazine.

Next, ABAP was bound to hemocyanin (KLH) derived from keyhole mussels via the aminobutyl group, and used as an immunogen. In addition to KLH, for example, proteins such as gamma globulin and bovine serum albumin obtained from chickens and the like may be used. Further, other amphetamine derivatives can be bound to proteins such as KLH via an alkyl group in the molecule. Binding of an amphetamine derivative such as ABAP to a protein can be performed by a usual method. That is, for example, N-succinimidyl 3- (2-pyridyldithio) -propionate (SPDP), tolylene-2,4-
Diisocyanate, glutaraldehyde, periodic acid,
1,5-difluoro-2,4-dinitrobenzene (DFDNB),
Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), N- (γ-maleimidobutyryloxy) succinimide (GMBS), N-
(Ε-maleimidocaproyloxy) succinimide (EMC
It can be performed using a conventional binding reagent such as S).

The immunogen thus obtained contains a structure with high chemical structural similarity to MA, which is the target antigen. This immunogen was injected into an animal to produce an antibody, and an ELISA using MA as a substance to be measured was performed using the obtained antibody.

The animals used for immunization can be any mammals, but mice were selected for ease of experiment. For the mouse strain, A / J had the highest immune response and was suitable.

As described above, a substance having a high chemical structure similar to the target antigen in a state where a functional group and a spacer have been introduced is used as a starting material, and an antigen derivative having a functional group and a spacer introduced thereto is converted into a protein via the functional group. If an immunogen prepared by binding to is used, the immunogen contains a structure with high chemical structural similarity to the target antigen. Therefore, when such an immunogen is injected into an animal, an antibody having a high affinity for the target antigen can be obtained from the serum of the animal.

Further, by fusing spleen cells of an animal sensitized with the immunogen with myeloma cells, a hybridoma producing a monoclonal antibody having a high affinity for the target antigen can be obtained. Hybridoma adjustment is performed by the method of Kohler and Milstein (Kohler et al., Nature, 256, 495 (1975), Kohler et.
al., Nature, 256, 495 (1975)).

Specifically, an immunogen in which ABAP and KLH are linked via the aminobutyl group of ABAP is injected into the body of an A / J mouse, and an antibody having a higher affinity for MA than ABMA is obtained from the serum. Could be obtained.

Furthermore, spleen cells of the mouse and mouse myeloma cells X63-Ag8.6653, one of 8-azaguanine resistant strains
Are fused in the presence of a fusion promoter such as polyethylene glycol, and the resulting fused cells are cultured in a HAT medium to produce a hybridoma that produces a monoclonal antibody having a higher affinity for MA than ABMA I was able to.

In addition, this hybridoma is cultured in a suitable medium,
A monoclonal antibody having higher affinity for MA than ABMA could be obtained from the supernatant.

Using antibodies and monoclonal antibodies with higher affinity for MA as compared to the obtained ABMA, ELISA using MA as the analyte was performed, compared to the case where antibodies obtained by the conventional method were used. Thus, the detection sensitivity of MA was improved about 1000 times. Therefore, these antibodies are extremely useful in the field of diagnosis, chemical analysis, and the like.

Example As an example of the present invention, MA, which is a stimulant and has a central nervous system stimulating effect, was used as an antigen of interest, and an antibody having high affinity for such antigen was produced. Hereinafter, the embodiment will be described in detail according to the operation procedure.

Example 1 First, a method for producing an antiserum will be described.

(1) Synthesis method of ABAP 2.00 g of AP, 4.17 g of N-bromobutylphthalimide (manufactured by Aldrich Chemical Company Inc.) and 3.14 g of sodium hydrogen carbonate were refluxed in 10 ml of benzene for 16 hours. After removing the solvent from the reaction solution, the reaction solution was purified using preparative thin-layer chromatography (TLC) using silica gel as a carrier (manufactured by Merck & Co., Inc.). As a developing solvent, a mixture of methanol and chloroform at a volume ratio of 5:95 was used after being saturated with ammonia. ABAP Rf value is 0.
It was 7. Extraction of ABAP in silica gel with methanol resulted in 1.59 g of N- (4-phthalimidylbutyl) amphetamine (PIBAP).

Dissolve 1.50 g of PIBAP in 5 ml of 95% ethanol and add 0.25 g
Was added and refluxed for 1.5 hours, whereupon a small amount of white precipitate was formed. After filtering again, the filtrate was
The mixture was acidified with 1N hydrochloric acid and the resulting white precipitate was removed. Further, when this solution was made alkaline with sodium hydroxide, an oil layer was separated. The oil layer was extracted with diethyl ether, and after distillation, purified by TLC under the same conditions as in the previous section. The target product reacts with the ninhydrin reagent to give a blue-violet color. Using this reaction, the target compound on TLC was confirmed. Finally, 0.75 g of N- (4-aminobutyl) amphetamine (ABAP) was obtained.

(2) Preparation of immunogen 19.1 mg of ABAP was dissolved in 200 μl of 0.5N hydrochloric acid and diluted with 4.8 ml of a 0.1 M phosphate buffer solution (pH 7.5). To this solution was added N-succinimidyl-3, a thiol group introduction reagent.
1 ml of-(2-pyridyldithio) propionate (SPDP) (Pharmacia KLB Biotechnology)
Was dissolved in ethanol, and the mixture was stirred and reacted for 30 minutes. To check the progress of the reaction, use TLC
Expanded on. The developing solvent used was the same as in the previous section. SPDP
Is identified on TLC as a black spot absorbing 254 nm ultraviolet light. In the reaction solution after stirring for 30 minutes, the black spot on TLC disappeared, and SPDP was completely reacted with ABAP. In this way, the combination of SPDP and ABAP (AP-
SPDP).

On the other hand, KLH was dissolved in a 0.1 M phosphate buffer (pH 7.0) containing 0.1 M NaCl to prepare a 2.2 mg / ml KLH solution. SP
A solution obtained by dissolving 51.6 mg of DP in 4.2 ml of ethanol was dropped into a KLH solution, and the mixture was stirred and reacted for 12 hours. Thereafter, unreacted unreacted components in the reaction solution were subjected to gel filtration using Sephadex G-25 (manufactured by Pharmacia LKB Biotechnology).
SPDP was separated to obtain a conjugate of KLH and SPDP (KLH-SPDP). Sephadex G-25 was packed in a column 4 cm in diameter and 50 cm in length and used at a flow rate of 6.9 ml per minute. Dissolve dithiothreitol in 0.1 M phosphate buffer,
l was added to KLH-SPDP for reduction. The mixed solution is
After gel filtration through a Sephadex G-25 column again under the same conditions as above, the AP-SPDP solution was gradually added to 106 ml of this solution. Thirty minutes later, gel filtration was performed using a Sephadex G-25 column under the same conditions as described above, and 14.3 AP molecules were introduced per KLH molecule. The conjugate of KLH and AP (AP-KLH) thus obtained was used as an immunogen.

(3) Immunization method The AP-KLH solution was diluted with a 0.1 M phosphate buffer solution (pH 7.0) containing 0.1 M NaCl to adjust the KLH concentration to 1 mg / ml.
Equal amounts of this AP-KLH solution and Complete Freund's adjuvant were mixed, and emulsified with a homogenizer.
Weekly mice were injected intraperitoneally. The injection volume was 100 μl per mouse. As the mouse strain, A / J was used as described above.

(4) Evaluation of antiserum by ELISA At the time point of 18 weeks after the immunization with AP-KLH, the serum of the mouse was collected. ELISA was performed using this serum to determine the measurement sensitivity when MA and ABMA were used as the substances to be measured. ELISA was performed under the following conditions.

96 wells (96 wells) made of polystyrene using a chemically conjugated product of BSA and ABMA (MA-BSA) in which 0.4 molecule of ABMA, which is an antigen derivative, is introduced for one molecule of bovine serum albumin (BSA) as a solid phase antigen It was adsorbed and fixed on an ELISA plate (Coaster Corporation). Antiserum collected from mice was washed with phosphate buffered serine (PB
It was diluted 10,000 times with S) and mixed 1: 1 with various concentrations of MA or ABMA solutions. This mixed solution was dispensed into a 96-well ELISA plate in an amount of 100 μl per well. After 3 hours, the mixed solution was removed, and the 96-well ELSIA plate was washed with PBS. By this operation, the antibody in the antiserum not bound to MA-BSA on the plate is removed. Next, an antibody having the binding ability to the mouse antibody contained in the antiserum was dispensed in the same amount as described above.
This antibody is a so-called POD-labeled antibody chemically bound to peroxidase (POD). After a lapse of 30 minutes, the POD-labeled antibody solution was removed, and the 96-well ELISA plate was washed again with PBS. By this operation, the POD-labeled antibody not bound to the mouse antibody bound to the solid phase antigen on the plate is removed. On the other hand, O-phenylenediamine (O
PD) 40mg with 0.1M phosphate citrate buffer (pH5.0) 10m
and mixed with ₄ μl of 30% H ₂ O ₂ . This mixed solution is washed with PBS after removing unbound POD-labeled antibody.
100 μl per well was dispensed into a well for ELISA plate. 1
After ~ 2 minutes, OPD was oxidized by the action of the POD of the POD-labeled antibody bound on the plate, and color development having a maximum at 492 nm was observed. After stopping the enzymatic reaction by injecting 25 μl per well of 4N sulfuric acid, the absorbance at 492 nm of the solution in each well was measured by a spectrophotometer. The measurement result is shown by a solid line in FIG. As comparative examples, MA and KLH synthesized in the same manner as AP-KLH were used.
The serum collected from a mouse immunized with the chemical conjugate (MA-KLH) at 18 weeks after the immunization was as follows:
The result of ELISA performed under the same conditions as above is shown by the dotted line in FIG.

In FIG. 2, the vertical axis indicates the relative absorbance at 492 nm, and indicates the enzyme activity. That is, it indicates the relative amount of the mouse antibody in the antiserum bound to the solid phase antigen, and if there is a decrease in the relative absorbance at a certain MA concentration, it can be said that the MA at that concentration can be detected. The horizontal axis is the antiserum used for measurement and MA or AB
Shows the concentration of MA or ABMA in the mixed solution of MA. The dotted line drawn beside represents 50% relative absorbance.

 Consider the results shown in FIG.

Curve a is a detection curve of MA by an antibody prepared by a conventional method,
Curve b is a detection curve of ABMA by an antibody obtained by a conventional method,
Curve c is a detection curve of MA by the antibody prepared by this method,
Curve d shows the detection curve of ABMA by the antibody prepared by this method.

First, with respect to the antiserum obtained by immunization with MA-KLH, the MA concentration at which the relative absorbance decreases to 50% is shown by curve a.
^-3.6M . Looking at curve B for ABMA, 10
^{At -5.2} M, the relative absorbance has been reduced to 50%. In other words, it can be said that ABMA inhibits the binding of the mouse antibody in the antiserum to the solid phase antigen about 50 times as strongly as MA. In other words, the antibodies obtained by MA-KLH immunization are more likely to produce ABM than MA.
Binds about 50 times stronger to A. On the other hand, considering that the solid phase antigen used for the ELISA is a chemically conjugated product of ABMA and BSA and contains the same structure as ABMA, the antibody obtained by MA-KLH immunization has a higher solid phase antigen than MA. Is expected to bind strongly to As mentioned above, the measurement principle of ELISA is
It utilizes the fact that the solid phase antigen and the antigen to be measured bind to the antibody competitively, resulting in the inhibition of the antibody binding to the solid phase antigen and the decrease in the amount of the antibody binding to the solid phase antigen. . If the antibody binds more strongly to the solid phase antigen, it is difficult for the antigen to be measured to inhibit the binding of the antibody to the solid phase antigen, resulting in a decrease in measurement sensitivity.

On the other hand, in the antiserum immunized with AP-KLH, the MA concentration when the relative absorbance decreased to 50% was 10
^{In the case of -6.5} M and ABMA, it was 10 ^-5.6 M from the curve d. In other words, the antibody obtained by immunization with AP-KLH, on the contrary to the case of immunization with MA-KLH, was about 50 times more effective against MA than the antigen derivative ABMA.
Binds twice as strongly. Therefore, a decrease in the measurement sensitivity as described above is not considered. In fact, in ELISA using an antiserum obtained by immunization with AP-KLH, ^10-6.5 M, which is a MA concentration at which the relative absorbance decreases to 50%, was determined using the antiserum obtained by immunization with MA-KLH. The concentration was about 1/1000 lower than that of

It should be noted that immunogens based on the above concept can be generally applied to the production of an antibody against a hapten having a molecular weight of 1,000 or less, other than MA shown in this example. Furthermore, an antibody produced from a hybridoma cell obtained by fusing the spleen cell and myeloma cell of the animal sensitized in this example,
So-called monoclonal antibodies naturally show the same properties as antisera. That is, it is possible to obtain a monoclonal antibody that binds most strongly to the hapten as the target antigen. Hereinafter, a method for producing a monoclonal antibody will be described.

Example 2 In Example 1, a monoclonal antibody was produced using a mouse in which production of an antibody that binds to MA more strongly than ABMA was confirmed. Hereinafter, the embodiment will be described following the procedure.

(1) Boosting of mice Seven weeks after immunization with AP-KLH, mice were boosted to enlarge the spleen. That is, AP
Dilute KLH to 1 mg / ml with PBS and add 100 μl / mouse
1 was injected intraperitoneally.

(2) Cell fusion Three days after the boost, mouse spleen cells were removed and fused with mouse myeloma cells X63-Ag8.653. The procedure will be described in detail below.

(I) Mice were killed and disinfected and moved to a clean bench. The following operations were all performed aseptically in a clean bench.

(Ii) The spleen of the mouse was excised and transferred onto a stainless mesh previously immersed in 5 ml of Ishikov modified Dulbecco's medium (IMDM) (manufactured by Sigma Chemical Company).
About 10 cuts were made in the spleen with scissors, and the spleen was carefully crushed using a glass rod for cell extraction. By this operation, the spleen cells are suspended in IMDM. After transferring the IMDM containing the spleen cells to a 15 ml centrifuge tube, the cells remaining on the mesh were further washed with IMDM to 5 ml, and this was also added to the centrifuge tube. The procedure up to (ix) below is to reduce the temperature of the cells to 4 ° C as much as possible.
The centrifuge was cooled so as to maintain the temperature, and an ice bath was used as needed.

(Iii) The suspension of spleen cells was centrifuged at 800 g for 7 minutes, and the supernatant was removed with an aspirator.

(Iv) To destroy red blood cells, which are unnecessary components, 10 ml of Tris buffer containing NH ₄ Cl was added, and the cells adhering to the bottom of the tube were stirred and loosened with a pipette. After leaving on ice for 5 to 10 minutes, the mixture was centrifuged at 800 g for 7 minutes, and the supernatant was removed with an aspirator.

(V) Hanks' balanced salt solution (manufactured by Osaka University Research Institute for Microbial Diseases)
After adding 10 ml and stirring and loosening the cells at the bottom of the centrifuge tube,
Leave on ice for 5-10 minutes. If the tissue fragments settle,
Only the cell suspension was transferred to another centrifuge tube so as not to remove this fragment. After centrifugation at 800 g for 7 minutes, the supernatant was removed with an aspirator.

(Vi) 10 ml of Hanks balanced salt solution was added again, and the cells at the bottom of the tube were stirred and loosened. After centrifugation at 800 g for 7 minutes, the supernatant was removed with an aspirator.

(Vii) 13-14 ml of Hanks' balanced salt solution was added, and the cells at the bottom of the tube were stirred and loosened.

(Viii) To 50 μl of the spleen cell suspension obtained in the previous section, 50 μl of a nigrosine solution as a cell staining solution was added, and about 1 minute later, the number of cells was determined using a hemocytometer. Cell count is 5.1x10 ⁶ cel
It was ls / ml.

(Ix) (The following operations are preferably performed alternately using the centrifugation time from the above (vi) to simultaneously count spleen cells and myeloma cells. Using a centrifuge set at a different temperature so as not to cool to below room temperature.) 45 ml of myeloma cells in the culture were transferred to a 50 ml centrifuge tube and centrifuged at 800 g for 7 minutes. The supernatant was removed with an aspirator.

(X) 10 ml of Hanks balanced salt solution was added, and the myeloma cells at the bottom of the tube were stirred and loosened. After centrifugation at 800 g for 7 minutes, the supernatant was removed with an aspirator. This operation was repeated twice.

(Xi) 13 ml of Hanks' balanced salt solution was added, and the myeloma cells at the bottom of the tube were stirred and loosened.

(Xii) The number of cells was measured in the same manner as in (viii). The number of cells was 1.5 × 10 ⁶ cells / ml.

(Xiii) 9 ml of the spleen cell suspension obtained in (viii) returned to room temperature and 6 ml of the myeloma cell suspension obtained in (xii) were mixed well, and centrifuged at 2000 g for 5 minutes. Was removed with an aspirator.

(Xiv) (The rest of the operation is about 40
C. in a water bath. ) Polyethylene glycol (PEG) with an average molecular weight of 1500 on the sedimented cells obtained in the previous section
0.5 ml was added over 1 minute with gentle stirring with a pipette. Stirring was continued for another 1.5 minutes.

(Xv) After adding 5 ml of IMDM at a rate of 1 ml / min, 5 ml of IMDM was added over the next minute. Finally, 10 ml of IMDM was added, followed by centrifugation at 1000 g for 7 minutes, and the supernatant containing PEG was completely removed with an aspirator.

(Xvi) 3 ml of the spleen cell suspension of (viii) was added as feeder cells to the sedimented cells obtained in the preceding paragraph, and HAT medium (Sigma Chemical) containing 10% fetal calf serum (FCS) was further added.
(Manufactured by Company) to make the total volume 23 ml. After loosening the cells with a pipette, 100 μl per well was dispensed into a 96-well plate for culture. The above operation was performed for two mice, and the cell suspension was dispensed to a total of six plates.

(Xvii) The plate was moved into a CO ₂ incubator and culture was started. The CO ₂ concentration in the incubator was kept at 5% and the temperature was kept at 37 ° C.

(Xviii) One day later, 100 μl of HAT medium was added per well, and the culture was continued for one week.

(3) Monocloning of antibody 1 after starting culture of hybridoma cells in HAT medium
The following operation was performed on the plate after one week.

(I) Collect 100 μl of culture supernatant per well from each well, and add PBS
To prepare a 2 to 10,000-fold dilution series, followed by ELISA using the dilution series. A solid phase antigen in which 0.4 molecules of MA are chemically bonded to one molecule of BSA (MA-BSA)
, And the substance to be measured was not added. Other procedures are the same as in the above-mentioned ELISA. In such an ELISA, the binding ability of the antibody in each culture supernatant to the solid phase antigen can be compared. That is, there is a high possibility that the culture supernatant showing a high absorbance at a higher dilution contains an antibody having a high affinity for the solid phase antigen. Some culture supernatants were selected from those with high absorbance, and the culture was continued only in the cells in the wells from which those culture supernatants were collected. The scale of the culture was increased as appropriate.

(Ii) More strict EL for the culture supernatant selected in the previous section
ISA was performed. The culture supernatant was appropriately diluted with PBS, and MA-BSA was used as a solid phase antigen. MA was added as the substance to be measured. Other operations are the same as in the above-described ELISA. M
Nineteen culture supernatants were selected from those with high measurement sensitivity of A, and the culture of the cells in the wells from which those culture supernatants were collected was continued.

(Iii) The cells that produced the selected 19 culture supernatants were each suspended in IMDM, and the number of cells was counted using nigrosine.

(Iv) Each cell suspension was diluted with HT medium (manufactured by Sigma Chemical Company), and cells were diluted to 1/100 μl.
Adjusted to be included.

(V) Excision of thoracic line from 5 week old mouse
It was suspended in HT medium and adjusted to contain 2 × 10 ⁵ thymocytes per 100 μl.

(Vi) The thymus cell suspension of (v) was dispensed into 10 96-well culture plates at 100 μl per well. And (iv)
Of the cell suspension was added in an amount of 100 μl per well.

(Vii) ELISA was performed on the culture supernatant while culturing was continued in a CO ₂ incubator. Conditions were the same as in (ii). With respect to the cells from which the culture supernatant with a high MA measurement sensitivity was obtained, the culture was continued while appropriately increasing the culture size.
As a result of continuing the selection by ELISA, 5 wells were finally selected. That is, five types of monoclonal antibody-producing cell lines were obtained. Place these cells in 200 ml IMDM per ml
Culture was continued until 5 × 10 ⁵ cells were included.

MA among 5 types of monoclonal cell producing cell lines
Cell line 2D55A, which produces a monoclonal antibody having the highest affinity for Escherichia coli, was deposited at the Patented Microorganisms Depositary Center under the Budapest Treaty at the Institute of Microorganisms and Industrial Technology of the Ministry of International Trade and Industry (Accession number: No.
No. 2564 (FERM BP-2564)).

(4) Storage of cells The culture solution of the finally selected cell line was transferred to a centrifuge tube, centrifuged at 800 g for 7 minutes, and the supernatant was removed with an aspirator. The cells at the bottom of the tube were suspended in a 9: 1 mixture of FCS and dimethylsulfoxide and adjusted to 5 × 10 ⁶ cells / ml. This suspension was frozen at -80 ° C and then transferred into liquid nitrogen for long-term storage.

(5) Antibody purification Protein A Sepharose 4B (Pharmacia LKB
Monoclonal antibodies were purified from the cell culture supernatant by affinity chromatography using Biotechnology). The purified monoclonal antibody is SD
By S polyacrylamide gel electrophoresis, the separation amount was about 50,
It was confirmed that the IgG was composed of 000 H chains and 20,000 L chains.

(6) Comparison of affinity of antibodies to various haptens ELISA was performed on purified monoclonal antibodies produced from 2D55A. MA-B for solid phase plateau
Using SA, measurement was performed for four types of haptens, MA, ABMA, AP, and ABAP. Other measurement conditions were the same as in the above-mentioned ELISA. When the concentration at which the relative absorbance at 492 nm was reduced to 50% was compared for the above four haptens, AP
Was the highest concentration, followed by ABMA, ABAP, and MA. To reduce the absorbance at 492 nm to 50%, AP
About 100 times the concentration of A was required. That is, AP-
By using KLH as an immunogen, it was proved that an antibody having the highest affinity for MA among the above four haptens could be produced.

[Brief description of the drawings]

FIG. 1 is a conceptual view of the process of the ELISA method, and FIG. 2 is a graph showing MAs obtained when ELISA was performed using the antisera obtained by the conventional method and the present method.
FIG. 7 is a diagram comparing the detection sensitivities of ABMA and ABMA. 1 ... microplate, 2 ... solid phase antigen, 3 ... antibody, 4 ... antigen, 5 ... antibody that binds to antibody 3, 6 ...
Enzyme chemically bound to Antibody 5

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Claims (12)

(57) [Claims] 1. An immunogen comprising methamphetamine and a protein bound to the methamphetamine via an alkyl chain as a spacer, wherein the N-methyl group in the methamphetamine molecule is replaced with a hydrogen atom. Using the corresponding amphetamine as a starting material, an alkyl chain and a functional group as a spacer are introduced into the primary amine of the amphetamine, and the affinity for methamphetamine obtained by binding to the protein via the alkyl chain and the functional group is obtained. An immunogen capable of inducing the production of antibodies having 2. The following structural formula The immunogen according to claim 1, wherein the immunogen is capable of inducing the production of an antibody having an affinity for methamphetamine, wherein the protein is bound to amphetamine via a terminal primary amine moiety of the amphetamine derivative represented by Here, n represents an integer. 3. The immunogen according to claim 2, wherein n is an integer from 1 to 10. 4. The immunogen according to claim 1, wherein the immunogen is capable of inducing the production of an antibody having an affinity for methamphetamine, in which aminobutylamphetamine is bound to a protein. 5. The immunogen according to claim 1, wherein the protein is any of hemocyanin derived from squash, gamma globulin derived from chicken, and bovine serum albumin. 6. An antibody having an affinity for the hapten obtained from the serum of an animal sensitized with the immunogen according to claim 1. 7. The antibody according to claim 6, wherein the immunogen is the immunogen according to claim 4. 8. A hybridoma which produces a monoclonal antibody having an affinity for the hapten, obtained by fusing spleen cells and myeloma cells of an animal sensitized with the immunogen according to claim 1. 9. The hybridoma according to claim 8, wherein spleen cells of an animal sensitized by the immunogen according to claim 4 are used. (10) Accession No. Micromachin Kenjo No. 2564 (FERM BP)
The hybridoma according to claim 8, which has (-2564). 11. A monoclonal antibody produced from the hybridoma according to claim 8. 12. The monoclonal antibody according to claim 11, which is an IgG comprising a heavy chain of about 50,000 and a light chain of about 20,000.