JPH0346562A - Immunization and immunoassay - Google Patents

Abstract

PURPOSE:To obtain high affinity by using a deriv. into which a carboxyl group is introduced by conjugating p-aminobenzoic acid with tetrahydrocannabinol and which is covalent-bonded to protein as an immunogen and using a mouse of an A/J system as an immune sensitized animal. CONSTITUTION:The spleen cells of the mouse (A/J system) immunized with the conjugate of the deriv. into which the carboxyl group is introduced by conjugating the p-aminobenzoic acid with the tetrahydrocannabinol or the analogous body thereof and the protein (hemocyanin derived from Fissurellidae or the gamma-globulin derived from chitin) as the immunogen and the cells derived from the myeloma of the mouse are fused and are cloned, by which the monoclonal antibody having the high affinity to the tetrahydrocannabinol and the analogous body thereof and the hybridoma cells to produce the antibody thereof are produced.

Description

Detailed Description of the Invention Industrial Application Field of the Invention (i) Immunization method for producing monoclonal antibodies, antiserum, etc. Antibody produced by this method and immunoassay for measuring the antigen-binding ability of this antibody Method The present invention further relates to a detection method using this monoclonal antibody.

BACKGROUND OF THE INVENTION Antibodies that specifically bind to a particular substance (antigen) are produced by injecting the antigen into an animal. If the antigen is a protein with a similar molecular weight, it is possible to inject the antigen as is.If it is a small molecule (hapten), it is necessary to inject an immunogen that has been linked to an appropriate protein and has a pseudo-high molecular weight. For example, antibodies against tetrahydrocannabinol or tetrahydrocannabinol analogs can also be developed using such methods.
Orman, L. J., Kink 0., Eherin M., Viall et al., Marks et al. (J, D., T.
eal, Elvyne J., Forman, L.
J.King.

Evelyn M, Piall and VoMark
s, J, Pharm, Pharmac.

(1975) 27, 465-472). However, the reported antibodies are polyclonal antibodies obtained by purifying the blood of immunized sheep. In addition, evaluation of the produced antibodies (an immunoassay method using an antigen labeled with a radioactive element is used).

Problems to be Solved by the Invention Conventional polyclonal antibodies use relatively large animals such as rabbits and sheep as immunization animals, and have the advantage that the entire manufacturing process is simple because they are obtained from their blood. Because the characteristics of the polyclonal antibodies obtained depend on each individual animal used, it is difficult to obtain antibodies with reproducible antigen-binding ability.

On the other hand, the safety of the developed evaluation method is also questionable because it uses radioactive elements.

The present invention ζ aims to solve the problems of the prior art.

Means for Solving the Problems The present invention (& In the immunization method for producing antibodies against tetrahydrocannabinol or its analogues, a carboxyl group is introduced by bonding p-aminobenzoic acid to tetrahydrocannabinol or its analogues. This immunization method is characterized by using a derivative covalently bound to a protein as an immunogen and using A/J strain mice as immunized animals.

That is, mice (A/J Monoclonal antibodies with high affinity for tetrahydrocannabinol or tetrahydrocannabinol analogs and hybridoma cells that produce the antibodies can be produced by fusing and cloning spleen cells of a mouse myeloma-derived cell line with mouse myeloma-derived cells.

In the present invention, the target antigen is tetrahydrocannabinol or a tetrahydrocannabinol analog,
All of these substances act as immunogens only when they are bound to macromolecules such as low molecular weight proteins. In this case, the protein (preferably one with good immune response) is a protein derived from chicken that is suitable in this respect.
Either globulin (CGG) or keyhole keyhole hemocyanin (KLH) is used. Furthermore, in order to create antibodies that specifically bind to tetrahydrocannabinol or tetrahydrocannabinol analogs, we investigated the structure of tetrahydrocannabinol or tetrahydrocannabinol analogs (especially the phenolic OH group).
Synthesize the immunogen while preserving as much as possible.

In addition, mice that are sensitized with an immunogen (those that produce antibodies with a higher titer are desirable). On the other hand, we used mice of the A/J strain, which is a suitable strain in this respect, to select or generate hybridoma cells. For antibody evaluation, an enzyme-linked immunosorbent assay is performed using a conjugate of tetrahydrocannabinol or a derivative of a tetrahydrocannabinol analog with a carboxyl group introduced as a solid-phase antigen and bovine serum albumin (BSA).

action Cells that produce immunoglobulins accumulate in the spleen.

Spleen cells themselves do not have the ability to proliferate, but by fusing with myeloma cells, it is possible to produce hybridoma cells that proliferate and produce antibodies. When a single hybridoma cell that produces an antibody with the highest affinity and has a high proliferative ability is selected (cloned) and cultured, a monoclonal antibody with high affinity is produced. Monoclonal antibodies have high affinity because they are antibodies of the same species. Furthermore, by culturing hybridoma cells, monoclonal antibodies with constant characteristics can be permanently provided.

Furthermore, by using an immunogen with a different protein portion, it is possible to obtain antibodies that accurately recognize only the target antigen and hybridoma cells that produce the antibodies, and it is also possible to evaluate the produced antibodies.

EXAMPLES Below, examples of the present invention will be described with reference to the drawings.

Hereinafter, the antibodies (antiserum and monoclonal antibodies) against tetrahydrocannabinol or tetrahydrocannabinol analogs of the present invention, the monoclonal antibody-producing hybridoma cells, and the binding ability of the antibodies to the antigen (tetrahydrocannabinol or tetrahydrocannabinol analogs) will be measured. The experimental methods for immunoassay are described below in order.

1: Preparation of immunogen Examples of immunogen synthesis are shown below.

(A) Isolation of Tetrahydrocannabinol (THC) Extract 5.2 g of tetrahydrocannabinol (THC) from 17.3 g of cannabis resin with petroleum ether.1. Using this energy as a solvent, 680 mg of TH
C was isolated from tetrahydrocannabinol (Due to the difference in the position of the double bond, △9-THC and △8-THC
The THC extracted here is △9-TH
The bottom of (B) tetrahydrocannabinol-2-p-azobenzoic acid (azoTHC-COOH), which was confirmed to be C. Using the THC isolated in section (A), the report by V, K., Marchie et al. First, 151 mg of p-aminobenzoic acid was added to INHC]14
10-Meanwhile, 312 mg of THC was dissolved in 19 ml of MeOH and 92 mg of sodium nitrite was gradually added to it.
This T was adjusted to pH 10-IL with INNaOH.
Add the previous p-aminobenzoic acid solution to the HC solution (about 10 p)
Then, adjust the pH with INHcI.
After adjusting the concentration to about 2, the solution extracted with dichloromethane was subjected to high performance liquid chromatography (HPLC).
Purification of zoTHC-COOH (C) Method for binding THC derivative and CGG azoTHC-
40 mg of C○OH was added to the solvent (pyridine: dioxane-1:
2) Dissolve 1-ethyl in 3 ml and stir.
After gradually adding 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (16.8 mg/1 ml phosphate buffer (0.1 M, PH7)), the CGG solution (30 ml) was left stirring for 3 hours. , 0.34%) was gradually added to the solution while stirring at 4°C, and further stirred at 4°C overnight.Then, the denatured protein was centrifuged (19000 rpm, 40ml).
After removal by gel filtration using Sephadex G-25 (manufactured by Pharmacia), azoTHC-CGG was obtained by separating the fraction showing absorption at 280 nm using Sephadex G-25 (manufactured by Pharmacia). Preparation azo THC-C
GG was diluted with PBS to make 6 ml of a 1 mg/m] solution Complete Freud's adjuvant (Wako Pure Chemical Industries H37R)
v) Or ζ Take 6 ml of each incomplete Freud's adjuvant (manufactured by Wako Pure Chemical Industries) while stirring well, and stir them with a homogenizer (10,00 rpm).
6ml of the previously prepared azo THC-CGG solution
In this example, △9-THC was used to generate the immunogen, which was combined with △8-THC and tetrahydrocannabinol analogues such as 11-hydroxy -△9-tetrahydrocannapine jl, 11-hydroxy-△
8-tetrahydrocannabinol, 8β hydroxy-
△9-tetrahydrocannabinol, 8β-hydroxy-△8-tetrahydrocannapine, cannabinol k, cannabidiol, cannabidiolic acid, cannabidichlor, cannabichromene, 8α-hydroxy-hexahydrocannabinol L/, 11-hydroxycannabinol,
Using exactly the same synthetic method for cannabinol-11-aldehyde acetate and 8-acetoxy-9-hydroxy-hexahydrocannabinol,
Immunogens for producing antibodies (antiserum, monoclonal antibodies) and antibody-producing hybridoma cells can be produced.

2: Method for producing monoclonal antibody-producing hybridoma cells and monoclonal antibodies Examples of production of monoclonal antibody-producing hybridoma cells and monoclonal antibodies are shown below.

(A) Immunization of mice Approximately 8 weeks after postnatal mice (A/J) 10 mice were injected with the complete Freud's adjuvant emulsion containing the immunogen prepared in Example 1 into their abdominal cavities in 100 μm increments, and after 21 days, the immunogen was injected again. Incomplete Freud's adjuvant containing immunogen was injected in 100 μm portions (B) Antibody production check 3 - Mice were injected 7 days after immunization with incomplete Freud's adjuvant containing immunogen, and were injected into the eye vein at 50 μm.
Collect 100 μl of blood into a centrifuge tube and keep the blood at 37°C.
After leaving it in a constant temperature bath for 10 minutes, centrifuge it to obtain serum.
When the serum was screened using the EIISA method (described later), the production of anti-tetrahydrocannabinol antibodies was confirmed. (C) Mouse boost (B) The titer was particularly high in the screening for section 2.
The immunogen was azoTHCCGG in PBS and boosted to enlarge the spleen of the mice.
(D) After 3 days of cell fusion boost, the spleen cells of the mice were removed by a known method using polyethylene glycol with an average molecular weight of 1.500. , mouse myeloma-derived cells (P3X63-Ag
8.653) to LN, 96-well plate 4 using spleen cells from the same mouse as feeder cells.
- Cultured on two plates in HAT medium containing 10% fetal bovine serum, and after 1 week, cultured in HAT medium containing 15% fetal bovine serum.
(E) Screening by cloning ELISA method was performed by replacing the medium with 1. X, Top 12 wells with high titers were selected Diluted to a concentration containing 1 cell per well (limiting dilution) lA 9
Dispense into 12 6-well microplates.1. 5 week old mice (Balb/c) as feeder cells
Promote the culture by increasing the size of the plate that stimulated initial proliferation using thymocytes. ELISA on the supernatant at the appropriate time.
Hybridoma cells showing a high titer against LTHC and good growth were screened repeatedly using the method, and hybridoma cells showing good growth were finally selected and incubated at 5 x lO' in 200 ml.
5 x 10' of finally selected hybridoma cells were cultured and flooded (F) until reaching a concentration of +nl.
e2: L-135 suspended in 1 ml of DMSO = 9:1 solution
5-(G) Monoclonal antibody was stored frozen at
A monoclonal antibody was purified from cell culture supernatant by affinity chromatography using A-3epharose CL-4B). This monoclonal antibody was analyzed by SDS polyacrylamide gel electrophoresis to determine the H chain with a molecular weight of approximately 50,000 and the L chain with a molecular weight of approximately 25,000. In this example, we used an immunogen using △9-THC, but this is not the case with △8-THC or a tetrahydrocannabinol analogue such as 11-hydroxy△ 9-tetrahydrocannabinol It/S 11-hydroxy-△8-tetrahydrocannabinol to 8β-hydroxy-△9-tetrahydrocannabinol I/, 8β-hydroxy-△8-tetrahydrocannabinol/lz,
cannabicyclo-k cannabidiol/lz, cannabidiolic acid cannabicyclo-/lz.

Immunogens using cannabicillomene, 8α-hydroxy-hexahydrocannabinol l/, 11-hydroxycannabinol, cannabinol-11-aldehyde a6-cetate and 8-acetoxy-9-hydroxyhexahydrocannabinol In these cases, monoclonal antibody-producing hybridoma cells can be similarly produced.

3: Evaluation of antibodies (antiserum, monoclonal antibodies) produced by immunoassay method 1.2, screening of antibody-producing hybridoma cells, and methods for detecting THC or THC analogs will be described below.

(A) Synthesis of solid-phase antigen azo 40 mg of THC-COOH was dissolved in 3 ml of solvent (pyridine:dioxane-1:2), and while stirring, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ( 16.8mg/
Add 1 ml of phosphate buffer (0.1 M, PH7)) to rounded corners, and then stir for 3 hours.
Gradually round the corners of the azOTHC-COOH solution while stirring at ℃.Stirring was continued at 4℃ for one night.Then, the denatured protein was centrifuged (19000 rpm, 40 m
1n), gel filtration with Sephadex G-25 (manufactured by Pharmacia), and az o
THC-BSA obtained (B) Coating of solid phase antigen The synthesized azo THC-BSA was mixed with Phospha containing 1% BSA and 0.04% sodium azide.
te-Buffered 5aline (BSA-P
Prepare the azoTHC-BSA concentration by diluting it with BS-Az) to a concentration of 0.001 mg/ml, and add it to a flooded microplate (polystyrene 96-well plate C03TA).
Inject this antigen solution at 100 μm/well into LA
(C) After removing the antigen solution by suction using a blocking aspirator, the BSA-P
1. Inject 250 μl/well of BS-Az and leave at room temperature for 1 hour. After that, wash with PBS three times, remove the remaining liquid with an aspirator, and 1° (D) Antibody reaction with antiblood? When evaluating culture supernatants, purified antibodies, or monoclonal 8-nal antibodies, use BSA-PBS-
When carrying out detection or inhibition experiments of tetrahydrocannabinol or tetrahydrocannabinol analogs, inject 100 μm/well of a solution (antiserum, monoclonal antibody purified on culture) appropriately diluted with Az, use an inhibitor solution (tetrahydrocannabinol). or tetrahydrocannabinol analog) 5
0 μl/well was injected and further 50 μl/well of monoclonal antibody solution was added After storing at room temperature for 3 hours, the antibody solution was removed with an aspirator L. Washed 3 times with PBS and the remaining PBS was removed with an aspirator (E) 2nd Antibody reaction 0.2 μg/ml goat-derived peroxidase-labeled anti-mouse gG antibody (KPL Cat, 141806 lo
Inject a BSA-PBS solution (second antibody solution) containing HLIO-5) at 25 μm/well and leave it for 30 minutes at room temperature. Remove the second antibody solution with an aspirator.
Wash 3 times with PBS and use an aspirator to remove remaining P.
Remove BS f.

(F) Color reaction and termination using peroxidase 9-0-Phenylenediamine (for biochemistry) 40 mg to 10
ml of citric acid-phosphate buffer (pH 5) and added 4 μl of 30% hydrogen peroxide immediately before use (
Substrate solution) was injected at 100 μl/well and left at room temperature for 10 minutes. After 10 minutes, 4N sulfuric acid was injected at 25 μm/well to stop the reaction. (G) Measurement at 492 nm using a Toyo Soda microplate reader.
Measure the absorbance with light f,.

By using this immunoassay method (inhibition method), it is possible to evaluate antibodies or use a very small amount of TH of 10-'M.
We were able to detect C (see drawing IGI).

Detection of THC by ELISA method (inhibition experiment).

The figure is a diagram showing the results of inhibition experiments using THC shown in Examples of the present invention (the vertical axis shows the absorbance at 492 nm CL, and the horizontal axis shows the concentration of 41 THC in logarithm).

It is also known that monoclonal antibodies (such as monoclonal antibodies) usually show affinity for substances that have a similar structure to immunogens.
HC analogs can be detected.

Below, the relative sensitivities of Δ8-THC and tetrahydrocannabinol analogs when performed using the same detection method as in this example are listed. Here, the relative sensitivity is △9-THC
The minimum detected concentration of each substance is shown when the minimum detected concentration of is set to 1. The higher the value, the higher the minimum detected concentration.

△8-THC5 11-human hydroxyl △9-detrahydrocannahi 9-nol, 3411-human hydroxyl △8-detrahydrocannahi 1-nol, 368β-human hydroxyl △9-detrahydrocannabinol, 248β- Human Roxy△
8-tetrahydrocannahinol, 32kannahi9nol,
52 Kannahishi All,
l 03 cannabiolic acid,
82 cannahicyclol,

85 kanna 7゛chromeene,
1028α-human, hexahuman, and cannahiol, 1
0811-Humanized xycannahynol, 6821 Cannahynol-11-aldehyde acetate
3058-Acetoxy-9-hydroxyhexahedral cannabinol 501
Effects of the Invention The present invention can provide a method for producing antibodies (antiserum, monoclonal antibodies) and monoclonal antibodies against tetrahydrocannabinol or tetrahydrocannabinol analogs with good reproducibility. Furthermore, it has become possible to provide a method for detecting tetrahydrocannabinol or a tetrahydrocannabinol analog using this monoclonal antibody. By using it as a phase antigen, it is possible to perform safe and accurate antibody evaluation.

[Brief explanation of drawings]

The figure is a graph showing the results of inhibition experiments using THC shown in Examples of the present invention.

Claims (11)

[Claims] (1) In an immunization method for producing antibodies against tetrahydrocannabinol or its analogs, a derivative in which a carboxyl group is introduced by bonding p-aminobenzoic acid to tetrahydrocannabinol or its analogs is covalently bonded to a protein. An immunization method characterized by using A/J strain mice as immunogens and immunizing animals. (2) In order to measure the binding ability of antibodies (antiserum or monoclonal antibodies) to tetrahydrocannabinol or its analogs, p-aminobenzoic acid is bound to tetrahydrocannabinol or its analogs as a solid-phase antigen. An immunoassay method characterized in that a derivative having a carboxyl group introduced therein is covalently bonded to bovine serum albumin (BSA). (3) The analogue is 11-hydroxy-△9-tetrahydrocannabinol, 11-hydroxy-△8-tetrahydrocannabinol, 8β-hydroxy-△9-tetrahydrocannabinol, 8β-hydroxy-△8-tetrahydrocannabinol , cannabinol, cannabidiol, cannabidiolic acid, cannabicyclol, cannabichromene, 8α-hydroxy-hexahydrocannabinol, 11-hydroxycannabinol, cannabinol-11-aldehyde acetate or 8-acetoxy-9-hydroxy-hexa The immunization method according to claim 1, wherein the immunization method is hydrocannabinol. (4) Analogs include 11-hydroxy-△9-tetrahydrocannabinol, 11-hydroxy-△8-tetrahydrocannabinol, 8β-hydroxy-△9-tetrahydrocannabinol, 8β-hydroxy-△8-tetrahydrocannabinol , cannabinol, cannabidiol, cannabidiolic acid, cannabicyclol, cannabichromene, 8α-hydroxy-hexahydrocannabinol, 11-hydroxycannabinol, cannabinol-11-aldehyde acetate or 8-acetoxy-9-hydroxy-hexa The immunoassay method according to claim 2, wherein the immunoassay method is hydrocannabinol. (5) The immunogen protein is hemocyanin (KLH) derived from keyhole limpet or γ-globulin (C) derived from chicken.
The immunization method according to claim 1, characterized in that the immunization method is GG). (6) The immunization method according to claim 1, wherein the covalent bond is an amide bond formed by using dichlorohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. (7) The immunoassay method according to claim 2, wherein the covalent bond is an amide bond formed by using dichlorohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. (8) An antibody produced by the immunization method according to claim 1. (9) A hybridoma cell, which is produced by fusing spleen cells and myeloma cells of an animal sensitized by the immunization method according to claim 1. (10) A monoclonal antibody obtained by purifying the culture supernatant of the hybridoma cell according to claim 9. (11) A detection method characterized by using the monoclonal antibody according to claim 10.