JPH02216459A - Antibody having high affinity with antigen being hapten - Google Patents

Abstract

PURPOSE:To detect an antigen of a substance to be measured with high sensitivity by using a substance having high similarity to an object antigen as a start substance for synthesizing an antigen derivative in a state that a functional group and a spacer are led in. CONSTITUTION:In a state that a functional group and a spacer are led in, a substance having high similarity on a chemical structure to an object antigen is used as a substance, and an antigen derivative obtained by leading the functional group and the spacer into this substance is coupled with protein through the functional group and an immunity source is manufactured. This immunity source contains a structure having high similarity on a chemical structure to the object antigen. Accordingly, when such an immunity source is injected into an animal body, an antibody having high affinity against the object antigen is obtained from a blood serum of an animal.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to improving the performance of antibodies that play the most important role in immunological detection methods useful in the field of medical diagnosis, chemical analysis, and the like. In particular, it relates to the production of antibodies that have high affinity for low-molecular compounds, ie, haptens, which cannot cause an immune response by themselves.

More specifically, the present invention relates to the production of antibodies and monoclonal antibodies that have a high affinity for methamphetamine, which is a type of stimulant that has a central nervous system stimulating effect.

BACKGROUND ART When detecting trace components in blood or specific components in the atmosphere, it is necessary to reliably detect a specific microscopic component from among a large number of impurities. For this purpose, immunological detection methods based on antibody reactions have been actively researched in recent years.

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

Here, the enzyme-linked immunononlubento assay (ELISA) method, which is a common EIA method, will be described. In FIG. 1, numeral 1 is a plastic microplate (such as Borisden) that nonspecifically adsorbs proteins. 2 is a solid-phase antigen in which an appropriate functional group is introduced into an antigen, which is a substance to be measured, and is chemically bonded to a protein through this, and is adsorbed on the microplate 1. 3 is an antibody that has the ability to bind to a non-analyte substance, and binds to the solid phase antigen 2 in equilibrium. That is, the antibody 3 is immobilized on the microplate 1 via the solid phase antigen 2. In this state, when antigen 4, which is the substance to be measured, is introduced, solid-phase antigen 2 and antigen 4, which is the substance to be measured, competitively bind to antibody 3. Therefore, a portion of the antibody 3 binds to the antigen 4, which is the substance to be measured, and as a result, a portion of the antibody 3 is not immobilized on the microplate 1. Naturally, the more antibodies 4 that are the substance to be measured, the more antibodies 3 that are not immobilized on the microplate 1. By washing here, antibodies 3 that are not immobilized on the microplate can be removed. After removing the antibody 3 that is not immobilized on the microplate I, the antibody 5 that has the ability to bind to the antibody 3 is added. However, antibody 5 is chemically bonded to enzyme 6 at a fixed ratio. After removing antibody 5 unbound to antibody 3 by washing, enzyme 6
Measure the activity of Since there is a positive correlation between the activity and the amount of enzyme G, the amount of enzyme G can be quantified by measuring the activity. If the amount of enzyme 6 is known, it is possible to retroactively quantify the amount of antibody 3 immobilized on microplate 1 via solid phase antigen 2, and further to quantify antigen 4, which is the substance to be measured. At this time, the first factor determining the detection sensitivity is the affinity of the antibody 3 for the antigen 4, which is the substance to be measured.

Antibodies are a type of protein produced by living organisms that have the property of selectively binding only to specific substances, that is, antigens. Antigens are broadly classified into two types based on the differences in the methods for producing antibodies that bind to them. One type is one that can produce antibodies by directly injecting it into an animal's body, and is usually a macromolecule with a molecular weight of about 100,000 yen or more. The other type has a relatively small molecular weight and can become an immunogen that induces antibody production only when chemically bonded to an appropriate protein. In particular, antigens such as the latter are called haptens. Regarding the method for producing an antibody against an antigen which is a hapten, for example, the following Bunyu (Haptens and Carriers, O. Makela and 1. J. T.

Separa, Handbook of Experimental Immunology Force Edicidon, vOl, 1.
Chap 3, :L Deity Rad by D,
M.

vair blackwell scientific
Publication Suns, Oxford, 198G,
Haptens and Carrlers, 0.
Kakela and 1, J, T, 5eppala
, Handbook of Experts+
ental I+wmu-nology 4th e
dl-Non, Vol. 1. Chap 3. ed
by D.

M, Welr, Blackvell 5clent1
f1c Publiflcatlons.

Oxford, 198G). That is, if a hapten has a functional group capable of chemical bonding, the functional group is used to bind to a protein to form an immunogen. If the target hapten does not have a functional group, a hapten derivative is synthesized by introducing an appropriate functional group such as an amino group, carboxyl group, or hydroxyl group and a spacer consisting of an alkyl chain with about 1 to 10 carbon atoms. Thereafter, the immunogen is generally obtained by binding it to an appropriate protein using a cross-linking reagent or the like. To give a more specific example, Takami et al. have reported on the production of antibodies using methamphetamine (MA), a type of stimulant, as a hapten antigen (Takami, Futada and Takahashi, Japanese Journal of Legal Medicine, Takaml et al. , F
ukuda and Takahashl, Jpn,
J, I Legal Med, 37(4), 4
17j1983). That is, by introducing an aminobutyl group into the secondary amine of MA, aminobutylmethamphetamine (
ABMA), which is combined with a protein and used as an immunogen.

Problems to be Solved by the Invention However, studies by the inventors have revealed that antibodies produced with the above immunogen have a higher affinity for ABMA, an antigen derivative, than for HA, the target antigen. Became. That is, antibodies obtained by conventional methods are only antibodies against antigen derivatives. Antigen derivatives have a structure different from that of the original target antigen due to the introduction of functional groups, spacers, etc., and this highlights the problem that it is difficult to create antibodies with high affinity for the target antigen. Ta.

Moreover, for this reason, when measuring the originally intended antigen as a analyte in the above-mentioned ELISA method using antibodies obtained by conventional methods, even if a large amount of the antigen, which is the analyte, is introduced, micro Since the amount of antibodies that are not immobilized on the plate does not increase, it becomes impossible to detect the antigen, which is the substance to be measured, with high sensitivity.

Means for Solving the Problems In order to solve the above problems, we considered using a substance highly similar to the target antigen with a functional group and a spacer introduced therein as a starting material for antigen derivative synthesis.

Specifically, a substance in which the alkyl group in the target antigen molecule is replaced with a hydrogen atom is used as a starting material, a functional group and a spacer capable of binding to a protein are introduced at the position of this hydrogen atom, and a protein is bonded via this functional group. It is conceivable to create an immunogen obtained by combining with

More specifically, the target antigen was methamphetamine (HA), which is a type of stimulant that has a central nervous system stimulant effect, and in order to produce antibodies with high affinity for such antigen, the N-alkyl group of MA was used as the target antigen. Aminobutylamphetamine (ABAP), which is an antigen derivative obtained by introducing an aminobutyl group as a spacer and a functional group capable of binding to proteins into amphetamine (AP), which has a structure in which a certain methyl group is replaced with a hydrogen atom, was used. APlABAP below
, MA, ABMA chemical structures are shown.

[AP] [ABMA] [AEIAP] [MA] It can be seen that there is a high degree of chemical structural similarity between MA and ABAP.

Other preferred antigen derivatives include amphetamine derivatives represented by the following structural formula (n represents an integer). n is an integer, and n is preferably an integer from 1 to 10. When n is 4, the following structural formula represents ABAP.

These amphetamine derivatives are produced by reacting AP with a tohaloalkyl phthalimide such as tobromobutylphthalimide to form N-(4-phthalimidyl alkyl).
It can be easily obtained by preparing amphetamine and then treating it with hydrazine.

Next, ABAP was combined with keyhole keyhole-derived hemocyanin (KLI()) via the aminobutyl group to serve as an immunogen.

In addition to KLH, proteins such as gamma globulin obtained from Ewatori and bovine serum albumin may also be used. Other amphetamine derivatives can also be linked to proteins such as KLI+ via alkyl groups in their molecules. Binding of an amphetamine derivative such as ABAP to a protein can be performed by a conventional method. That is, for example, N-succiimidyl 3-
(2-pyridyldithio)-propionate (SPDP)
, tolylene-2,4-diisocyanate, glutaraldehyde, periodic acid, l,5-difluoro-2,4-dinitrobenzene (DFDNI3), succinimidyl4
Common coupling reagents such as -(N-maleimidomethyl)cyclohexane-1-carboxylate CSMCC), N-(γ-maleimidobutyryloxy)succinimide (CMBS), N-(ε-maleimidocaproyloxy)succinimide (EMCS) It can be implemented using

The immunogen thus obtained contains a structure highly similar in chemical structure to H&, which is the target antigen.

This immunogen is injected into the animal body to produce antibodies, and the obtained antibodies are used for ELIS using MA as the analyte.
I did A.

Although any mammalian animal can be used for immunization, mice were selected for ease of experimentation. Among the mouse strains, A/J had the highest immune response and was the most suitable strain.

Action As described above, a substance with high chemical structure similarity to the target antigen with a functional group and spacer introduced therein is used as a starting material, and an antigen derivative with the functional group and spacer introduced therein is introduced into the protein via the functional group. When an immunogen prepared by binding with a target antigen is used, the immunogen contains a structure with high chemical structural similarity to the target antigen. Therefore, by injecting such an immunogen into an animal's body, antibodies with high affinity for the target antigen can be obtained from the animal's serum.

Furthermore, by fusing myeloma cells with spleen cells of an animal sensitized with the above immunogen, hybridomas that produce monoclonal antibodies with high affinity for the target antigen can be obtained. Hybridomas are prepared using the Köhler and Milstein method (Köhler et al.
Earl, Neich + -25G, 495 (1975),
Kohler et al., Nature, 25
G, 495 (1975)).

Specifically, an immunogen in which ABAP and KLII are combined via the aminobutyl group of MuRAP is injected into the body of A/J strain mice, and the serum has a higher affinity for HA than ARIA. We were able to obtain antibodies.

Furthermore, spleen cells of the mouse and mouse myeloma cells XG3-mug8.6, which is one of the 8-azaguanine-resistant strains, were
53 in the presence of a fusion promoter such as polyethylene glycol, and the resulting fused cells were cultured in flAT medium to generate hybridomas that produce monoclonal antibodies that have a higher affinity for MA than for muBMA. I was able to create one.

Furthermore, this hybridoma was cultured in an appropriate medium, and a monoclonal antibody having a higher affinity for HA than ABMA could be obtained from the supernatant.

When performing ELISA using MA as the analyte using an antibody and a monoclonal antibody that have a higher affinity for MA than the obtained ABilA, it was found that ELISA was performed using antibodies obtained by conventional methods. As a result, the detection sensitivity of MA was improved by about 1000 times. Therefore, these antibodies are extremely useful in the diagnostic field, chemical analysis, and the like.

EXAMPLE As an example of the present invention, HA, which is a stimulant having a central nervous system stimulating effect, was used as a target antigen, and an antibody having high affinity for the antigen was prepared.

Hereinafter, a detailed explanation of the embodiment will be given according to the operating procedure.

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

(1) ABAP synthesis method AP 2.00g, N-bromobutylphthalimide (Aldrich Chemical Company Inc.,
4.17 g of sodium bicarbonate and 3.14 g of sodium bicarbonate were refluxed in 10 sl of benzene for 16 hours. After removing the solvent from the reaction solution, preparative thin layer chromatography (TLC) using silica gel as a carrier (Merck & C
ozInc, Inc.). The developing solvent used was a mixture of methanol and chloroform in a volume ratio of 5=85, which was saturated with ammonia. The Rf value of ABAP was 0.7. 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% edanol and add 0.25 g of hydrazine l-hydrate.
When the mixture was refluxed for 5 hours, a small amount of white precipitate was formed. After filtration again, the filtrate was acidified with IN hydrochloric acid to remove the white precipitate that formed. Furthermore, when this solution was made alkaline with sodium hydroxide, an oil layer was separated. The oil layer was extracted with diethyl ether, distilled off, and purified by TLC under the same conditions as in the previous section. The target product reacts with the ninhydrin reagent and exhibits a blue-purple color. Using this reaction, the target product on TLC was confirmed. Finally, 0.75 g of N-(4-aminobutyl)amphetamine (ABAP) was obtained.

(2) Immunogen preparation method ABAP 19.1B was dissolved in 2011 μl of 0.5N hydrochloric acid, and 0°IM phosphate buffer 1-solution (pH 7,5
) Diluted with 4.8 ml. To this solution, a thiol group-introducing reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) (manufactured by Pharmacia LKB Biotechnology), was added at 12°5 m
A solution of 11 g of ethanol was added thereto, and the mixture was stirred for 30 minutes to react. In order to confirm the progress of the reaction, the reaction solution was developed on TLC. The developing solvent used was the same as in the previous section. 5PDP is confirmed on TLC as a black spot that absorbs 254 nm ultraviolet light. In the reaction solution that had been stirred for 30 minutes, the black spot on TLC had disappeared, indicating that 5PDP had completely reacted with ABAP. In this way, a conjugate of 5PDP and ABAP (AP-5P
I) P) was obtained.

On the other hand, KLH was 0.1M containing 0.1M NaCl.
Dissolved in phosphoric acid (pH 7.0), 2.2
A mg/ml KLH solution was prepared. 5PDP 51.
KLH
It was added dropwise to the solution and stirred for 12 hours to react. Thereafter, unreacted 5PDP in the reaction solution was separated by gel filtration using Sephadex G-25 (manufactured by Pharmacia LKB Biotechnology) to obtain a conjugate of KLII and 5PTIP (KLH-SPDP). Sephadex G
-25 was packed in a column with a diameter of 4 cm and a length of 50 cm.
A flow rate of 6.9011 per minute was used. Dithiothreitol was dissolved in O.1M phosphate buffer and this solution 1.
8 ml was added to KL[l-5PDP to perform reduction. The mixed solution was treated with Sephadex G again under the same conditions as above.
After gel filtration with 25 columns, 10 BIIll of this solution
The AP-SPDP solution was gradually added to the solution. After 30 minutes, gel filtration was performed using a Sephadex G-25 column under the same conditions as above.
4.3 molecules were introduced. KL obtained in this way
) A conjugate of I and AP (AP-KLH) was used as an immunogen.

(3) Immunization method AP-KLII solution was 0. 0.1 containing IM NaCl
M diluted with phosphate buffer 1-solution (pH 7,0), K
The LH'a level was adjusted to Img/ml. This AP-KL
) I solution and complete Freund's adjuvant were mixed in equal amounts and emulsified using a homogenizer, and the mixture was injected into the abdominal cavity of an 8-week-old mouse. The injection volume was 100 μl per mouse. Note that the mouse strain A/J was used as described above.

(4) Evaluation of antiserum by ELISA AP-KL)f
The serum of the mice was collected 18 weeks after immunization with the following. ELiSA was performed using this serum, and M
The measurement sensitivity was determined when A and ABMA were used as the 11111 constant substances. ELISA was performed under the following conditions.

A chemical conjugate of USA and ABMA (MA-BSA) into which 0.4 molecules of the antigen derivative AEtMA were introduced per molecule of bovine serum albumin (BSA) was used as a solid-phase antigen, and a 96-hole tube made of polystyrene ( 98vell)
It was fixed by adsorption on an ELISA plate (manufactured by Coaster Corporation). The antiserum collected from mice was diluted with phosphate buffered arteceline (PBS) at 10.0%.
The solution was diluted 1:00 and mixed with MA or ABMA solutions of various concentrations in Hl. Apply this mixed solution to a 96-well ELISA plate at 100μ per well! Dispensed. After 3 hours, remove the mixed solution and place the 96-well ELISA plate on
Washed with BS.

By this operation, antibodies in the antiserum that are not bound to MA-BSA on the plate are removed. Then, the same amount of an antibody capable of binding to the mouse antibody contained in the antiserum as above was dispensed. This antibody is chemically bonded to peroxidase (POD) and is a so-called POD-labeled antibody. After 30 minutes, remove the POD-labeled antibody solution and incubate with PBS again.
The 6-well ELISA plate was washed. By this operation, the POD-labeled antibody that is not bound to the mouse antibody bound to the solid-phase antigen on the plate is removed. On the other hand, 0-phenylenediamine (OPD), a substrate of POD, 40Ing
0.1M phosphate citrate buffer (pH 5,0)
Dissolved in IOn+I and mixed with 4 μm of 30% H2O2. After removing unbound POD-labeled antibody, this mixed solution was added to a 96-well E1 IsA plate that had been washed with PBS. 100 μm was dispensed per hole. After 1 to 2 minutes, O
The PD was oxidized and a color with a maximum at 492 nm was observed. After stopping the enzyme reaction by dispensing 25 μm of 4N sulfuric acid per hole, 492 μm of the solution in each chamber was measured using a spectrophotometer.
The absorbance at nm was measured. The measurement results are shown in solid lines in Figure 2.

As a comparative example, serum collected 18 weeks after immunization from mice immunized with a chemical conjugate of MA and KLII (HA-KLH) synthesized in the same manner as AP-KL11 was tested. The results of ELISA conducted under the same conditions are shown in FIG. 2 by dotted lines.

In FIG. 2, the vertical axis is the relative absorbance of 492nI11, indicating the enzyme activity. That is, it shows the relative amount of mouse antibody in the antiserum bound to the solid-phase antigen, and it shows the relative amount of mouse antibody in the antiserum bound to the solid-phase antigen, and for a given MA?
If there is a decrease in relative absorbance at J1 degree, the MA at that concentration
can be said to be detectable. The horizontal axis represents MA or AB of the mixed solution of antiserum and MA or ABMA used for measurement.
The concentration of MA is shown. The dotted line drawn horizontally indicates relative absorbance of 50%.
represents.

Let's consider the results shown in Figure 2.

Curve a is a detection curve for one person using an antibody prepared using a conventional method;
The curves are the detection curve of ABMA using the antibody obtained by the conventional method, the curve C is the detection curve of A using the antibody produced by the present method, and the curve d is the ABMA detection curve using the antibody produced by the present method.
The detection curve is shown.

*About the antiserum obtained by immunization with fMA-KL■,
Looking at the MA concentration at which the relative absorbance decreased by 50% in curve a, it was 10-3.6M. Regarding ABMA, when looking at curve B, the relative absorbance decreases to 50% at 10-6.2M.

That is, it can be said that ABMA inhibits the binding of mouse antibodies in antiserum to solid-phase antigen about 50 times more strongly than MA. In other words, antibodies obtained by HA-KLH immunization bind approximately 50 times more strongly to AHMA than to MA. On the other hand, the solid phase antigens used in ELISA were ABMA and B.
Considering that it is a chemical conjugate of SA and contains the same structure as ABMA, it is expected that the antibody obtained by MA-KLH immunization will bind more strongly to the solid-phase antigen than MA. As mentioned above, the measurement principle of ELISA is
This method utilizes the fact that the solid-phase antigen and the antigen to be measured competitively bind to the antibody, which inhibits the binding of the antibody to the solid-phase antigen and reduces the amount of antibody binding to the solid-phase antigen. . If the antibody binds more strongly to the solid-phase antigen, the binding of the antibody to the solid-phase antigen by the antigen, which is the analyte, will be inhibited.
<, resulting in a decrease in measurement sensitivity.

On the other hand, for the antiserum immunized with AP-KLH, when the relative absorbance decreased to 50%, the A concentration was on the curve C, IP'M1ABIIA, d, and 10-'M.

In other words, the antibodies obtained by immunization with AP-KLII are HA
-Contrary to the case of immunization with KLH, antigen derivative ABMA
It binds about 50 times more strongly to MA than to MA. Therefore, the decrease in measurement sensitivity as described above is unlikely. In fact, using antiserum obtained from immunization with AP-KLH,
In LISA, the M and A concentration at which the relative absorbance decreases by 50%, 10-'', is about 1000 times lower than when using the antiserum obtained by immunization with HA-KLH. there were.

In addition, the immunogen based on the above-mentioned concept can be generally applied to MAs other than MA shown in this example when producing antibodies against haptens with a molecular weight of 100 or less. Furthermore, antibodies produced from hybridoma cells obtained by fusing pancreatic cells and myeloma cells of the animals sensitized in this example, so-called monoclonal antibodies, naturally exhibit properties similar to those of antiserum.

That is, it is possible to obtain a monoclonal antibody that binds most strongly to the target antigen, hapten. The method for producing monoclonal antibodies will be described below.

Example 2 In Example 1, monoclonal antibodies were produced using mice that were confirmed to produce antibodies that bind more strongly to HA than ABMA. An example will be explained step by step below.

(1) Boosting of mice Seven weeks after immunization with AP-KLH, mice were boosted to enlarge their spleens. That is,
AP-KLH was diluted to 11g/ml with PBS and 100 μl per mouse was injected intraperitoneally.

(2) Mouse pancreatic cells were taken out at the third molar after cell fusion boost and fused with mouse myeloma cells X83-Ag3.653.

The steps are detailed below.

(1) After sacrificing and disinfecting the mouse, it was moved into a clean bench. All of the following operations were performed aseptically in a clean bench.

The spleen of a (+S) mouse was removed and transferred onto a stainless steel mesh that had been previously immersed in 51 Ischikov's modified Dulbecco's medium (IMDM) (manufactured by Sigma Chemical Company). Approximately 10 incisions were made in the spleen using scissors, and the spleen was carefully crushed using a glass rod for removing cells. By this operation, pancreatic cells are targetedly suspended in the IMD. After transferring the IMDM in which pancreatic cells were suspended to a 15ml centrifuge tube, the cells remaining on the mesh were further transferred to 51 IMDMs.
This was also added to the centrifuge tube. In the following operations up to (lx), the centrifuge was cooled and a water bath was used as needed to maintain the cell temperature at 4°C as much as possible.

(Eye 1) #The suspension of visceral cells was centrifuged at 800 g for 7 minutes, and the supernatant was removed using an aspirator.

(1v) NH4 to destroy red blood cells, which are unnecessary components.
10+gl of Tris buffer containing Cl was added, and the cells adhering to the bottom of the tube were stirred with a pipette to loosen them.

After being left on ice for 5 to 10 minutes, centrifugation was performed at 800 g for 7 minutes, and the supernatant was removed using an aspirator.

(y) Hanks Balanced Salt Solution (Osaka University Research Institute for Microbial Diseases Ii)
l) 10 ml was added, the cells at the bottom of the centrifuge tube were stirred to loosen them, and then left on ice for 5 to 10 minutes. If tissue fragments precipitated, only the cell suspension was transferred to another centrifuge tube so as not to remove the fragments. Centrifugation was performed at 800 g for 7 minutes, and the supernatant was removed using an aspirator.

(vl) 10 ml of Hank's balanced salt solution was added again, and the cells at the bottom of the tube were stirred to loosen them. Centrifugation was performed at 800 g for 7 minutes, and the supernatant was removed using an aspirator.

(vll) Add 13 to 14 ml of Hanks balanced salt solution,
The cells at the bottom of the tube were stirred to loosen them.

(50 μl of nigrosine solution, which is a cell staining solution, was added to 50 μl of the spleen cell suspension obtained in the previous section, and after about 1 minute, the number of cells was determined using a hemocytometer. The number of cells was 5.
, 1xlO'cel Is/ml.

(lx) (The following operations are preferably performed alternately using the centrifugation time from the above (vl) so that pancreatic cells and myeloma cells are counted at the same time.

Furthermore, in the subsequent operations, centrifuges with different temperature settings were used to avoid cooling the myeloma cells below room temperature.

) 45 IIll of myeloma cells in 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) Hank's Balanced Salt Solution 101 was added, and the myeloma cells at the bottom of the tube were stirred to loosen them. After centrifugation at 800g for 7 minutes, the supernatant was removed with an aspirator. This operation was repeated twice.

(xl) 13al of Hank's balanced salt solution was added, and the myeloma cells at the bottom of the tube were stirred to loosen them.

(Xth item) The number of cells was measured in the same manner as in CV item 1). The number of cells is 1.5xlO'cel Is/
It was n+I.

(xllf) Return the spleen cell suspension obtained in (vm) to room temperature, mix well with 11 vol of myeloma cell suspension obtained in (xl), centrifuge at 2000g for 5 minutes, and remove the upper groove. Removed with an aspirator.

(xlv) (The subsequent operations were performed in a water bath at approximately 40°C placed in a clean bench.) Polyethylene gel IJ:L (PEG ), 0.5 ml was added over 1 minute with warm stirring using a pipette. Another 1.5
Stirring was continued for a minute.

(xv) Furthermore, IMDM at a rate of 1ml/win 51
After the addition, IMDI 51 was added over the next 1 minute. Finally, after adding [MDM loml], centrifugation was performed at 1000 g for 7 minutes, and the supernatant containing PEG was completely removed using an aspirator.

(xvl) Add 31 spleen cell suspension (V 1) as feeder cells to the sedimented cells obtained in the previous section, and then add 31 spleen cell suspension as feeder cells.
I (AT medium (Sigma Chemical Company) containing 0% fetal calf serum (FCS) was added to bring the total volume to 23 ml.
And so. After loosening the cells with a pipette, place them in a 96-well plate for culture! A 100 μm portion was dispensed per hole. Perform the above procedure for two mice, and add cells to a total of 6 plates! ! ! The suspension was dispensed.

(XYll) The plate was moved into a CO2 incubator and culture was started. co2tM in incubator
The temperature was maintained at 5% and 37°C.

(xvill) 1 day later, 100 μl HA per lx
T medium was added and culture was continued for one week.

(3) Monocloning of antibodies 1 after the start of culturing of hybridoma cells in HAT medium
The following operations were performed on the plates after a week had passed.

(1) Collect 100 μm of culture supernatant from each person per hole,
After diluting with PBS to prepare a 2- to 10,000-fold dilution series, ELISA was performed using this. As a solid-phase antigen, one in which 4 molecules of MAO were chemically bound to one molecule of BSA (HA-BSA) was used, and no substance to be measured was added.

Other operating methods are the same as in the ELISA described above. In such an ELISA, the binding ability of antibodies in each culture supernatant to a solid-phase antigen can be compared. That is,
In the culture supernatant, which shows high absorbance at higher dilutions,
It is likely to contain antibodies with high affinity for solid-phase antigens. Several culture supernatants with high absorbance were selected, and only the large and medium cells from which these culture supernatants were collected were continued to be cultured.

The scale of culture was increased as appropriate.

(11) A more rigorous ELISA was performed on the culture supernatants selected in the previous section. The culture supernatant was appropriately diluted with PBS, and MA-USA was used as a solid phase antigen. HA was added as a substance to be measured. For other operations, use the EL mentioned above.
It is similar to ISA. 1 from the one with the highest MA measurement sensitivity
The culture supernatants of No. 3 were selected, and the culture of the large medium cells from which these culture supernatants were collected was continued.

(iii) Selected! The cells that produced the culture supernatant of No. 9 were each suspended in DM and the number of cells was counted using nigrosine.

(1v) Transfer each cell suspension to IIT medium (Sigma
(manufactured by Chemical Company) to make cells 100 μm thick.
Adjusted to include one item per person.

(Y) The thymus was removed from a 5-week-old mouse, and the thymocytes were suspended in ■T medium so that 2×10i thymocytes were contained per 100 μm.

, (Yl) The thymocyte suspension of (Y) was divided into 10 96
101) μl was dispensed per 1 hole into a well culture plate. Additionally, add 100μ of cell suspension (1v) per l well.
l added.

(vll) While continuing the culture in a CO2 incubator, ELISA of the culture supernatant was performed. The condition is (eyes)
Same as section. For cells from which culture supernatants with high HA measurement sensitivity were obtained, culture was continued while increasing the culture size as appropriate. As a result of continued selection by ELISA, 5 wells were finally selected. That is, five types of monoclonal antibody-producing cell lines were obtained. These cells are 200m
Culture was continued until 5xlO' cells per 11 cells were contained in 1 IMDM.

Among the 5m monoclonal cell production cell lines, MA
The cell line 2D55A, which produces monoclonal antibodies with the highest affinity for No. 2564 (FERMBP-2564)).

(4) Preservation 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 solution containing 3:1 of FCS and dimethyl sulfoxide, and 5X per 11
The suspension was adjusted to contain 1G" cells. The suspension was frozen at -80°C and then transferred to liquid nitrogen for long-term storage.

(5) Purification of antibodies Protein A Sepharose 4B (Pharmacia LKB
Monoclonal antibodies were purified from the cell culture supernatant by affinity chromatography using Biotechnology Co., Ltd.). The purified monoclonal antibody was separated by SOS polyacrylamide gel electrophoresis into two chains of approximately so, ooo and 20. It was confirmed that it was IgG consisting of the short chain of GOO.

(6) Comparison of affinity of antibodies for various haptens Among the purified monoclonal antibodies, ELISA was performed on those produced from 2D55A. MA-BSA was used as the solid phase antigen, and Ayu, ABMAlAP, AB
Measurements were performed on four types of haptens of AP. Other measurement conditions were the same as those for the above-mentioned ELISA. 492nm
When the concentration at which the relative absorbance of HA decreased to 50% was compared for the above four haptens, AP had the highest concentration, followed by ABMA, ABAP, and HA. Approximately 100 times the concentration of AP was required compared to HA to reduce the absorbance at 492 nm by 50%. That is, it was demonstrated that by using AP-KLII as an immunogen, an antibody having the highest affinity for 11A among the four types of haptens described above could be produced.

FIG. 3 is a diagram comparing the detection sensitivity of Nimu and MuBMA when ELISA was performed using antiserum obtained by the present method.

I...Φ microplate, 2... solid phase antigen, 3...
・Antibody, 4...antigen, 5...antibody that binds to working antibody 3, 6...enzyme chemically bound to fist antibody 5, name of agent, patent attorney Shigetaka Awano, and 1 other person pot diagram

Claims (13)

[Claims] (1) An immunogen consisting of a hapten and a protein bound to the hapten via an alkyl chain as a spacer, starting from a substance corresponding to a compound in which the alkyl group in the hapten molecule is replaced with a hydrogen atom. The immunogen obtained by introducing an alkyl chain and a functional group as a spacer into the position corresponding to the hydrogen atom of this starting material and binding it to the protein via this alkyl chain and functional group becomes a hapten. An immunogen that has a chemical structure that is highly similar to that of an immunogen. (2) The hapten is methamphetamine, and amphetamine corresponding to a compound in which the N-methyl group in the molecule of this hapten is replaced with a hydrogen atom is used as a starting material, and an alkyl chain and a functional group as a spacer are attached to the primary amine of amphetamine. 2. The immunogen according to claim 1, which has the ability to have high affinity for methamphetamine, which is obtained by introducing and binding to the protein via the alkyl chain and functional group. (3) Production of an antibody with high affinity for methamphetamine by binding amphetamine and protein via the terminal primary amine moiety of an amphetamine derivative represented by the following structural formula ▲ Numerical formula, chemical formula, table, etc. ▼ The immunogen according to claim 1, which is capable of inducing. However, n
indicates an integer. (4) The immunogen according to claim 3, wherein n is an integer from 1 to 10. (5) The immunogen according to claim 1, which is a combination of aminobutylamphetamine and a protein and is capable of inducing the production of an antibody having high affinity for methamphetamine. (6) The immunogen according to claim 1, wherein the protein is any one of hemocyanin derived from keyhole limpet, gamma globulin derived from Ewatori, and bovine serum albumin. (7) An antibody having high affinity for the hapten obtained from the serum of an animal sensitized with the immunogen according to claim 1. (8) The antibody according to claim 7, wherein the immunogen is the immunogen according to claim 5. (9) A hybridoma which produces a monoclonal antibody having high affinity for the hapten, which is obtained by fusing spleen cells and myeloma cells of an animal sensitized with the immunogen according to claim 1. (10) The hybridoma according to claim 9, which uses spleen cells of an animal sensitized with the immunogen according to claim 5. (11) Accession number FERM No. 2564 (FERM
10. The hybridoma according to claim 9, which has BP-2564). (12) A monoclonal antibody produced by the hybridoma according to claim 9. (13) H chain with a molecular weight of about 50,000 and a molecular weight of about 20,000
13. The monoclonal antibody according to claim 12, which is IgG consisting of an L chain of.