JP6493786B2 - Immunogen, method for producing antibody using the same, and monoclonal antibody thereof - Google Patents

Description

  The present invention relates to antibodies necessary for detecting synthetic cannabinoids, methods for producing the same, and immunogens in industrial fields such as legal management and health management.

  In order to detect synthetic cannabinoids, it is usually required to perform detection work quickly and accurately. In addition, in the detection of the above synthetic cannabinoids, it is also required to analyze trace components.

  Conventionally, as a method for detecting synthetic cannabinoids, there have been gas chromatograph mass spectrometry (GC-MS), liquid chromatograph mass spectrometry (LC-MS), or a measurement method using an ionizer / collector device disclosed in Patent Document 1. .

Special table 2007-515619

  The sensitivity of GC-MS and LC-MS, which are conventional methods, is on the order of about ppm. Further, when impurities are present, there is a problem that the sensitivity is significantly lowered.

  Therefore, it is necessary to remove a certain amount of impurities as a pretreatment, and as a result, there is a problem that the measurement time also takes a long time (30 minutes or more).

  In order to solve the above-mentioned conventional problems, the immunogen (synthetic cannabinoid immunogen) of the present invention is obtained by binding a compound having the structure of the structural formula (Formula 1) to keyhole limpet hemocyanin, which is a carrier protein. is there.

  Furthermore, it is possible to provide a method for producing a monoclonal antibody-producing cell line that can immunize an animal (mouse, rabbit, etc.) with the immunogen and produce a specific antibody.

  In addition, according to the method for producing a monoclonal antibody-producing cell line, any one of the following monoclonal antibody-producing cell lines, which is a cell line that produces a monoclonal antibody capable of binding to the compound, can be provided. A cell line deposited as deposit number NITE BP-01955, a cell line deposited as deposit number NITE BP-0195, and a cell line deposited as deposit number NITE BP-01957.

  Furthermore, a monoclonal antibody that reacts with the compound produced by the cell line described above.

  Higher-performance antibodies can be produced than the synthetic cannabinoid derivatives and antibody production methods of the present invention. In addition, synthetic cannabinoids can be rapidly and accurately analyzed by using the above-described antibodies.

Evaluation of binding ability of three types of monoclonal antibodies by ELISA

  Embodiments of the present invention will be described below with reference to the drawings.

  Hereinafter, the present invention will be described more specifically with reference to examples.

  Hereinafter, a method for producing a synthetic cannabinoid derivative possessing a carboxyl group and a method for detecting a synthetic cannabinoid using this antibody will be described.

(1) Synthesis of synthetic cannabinoid derivative 2.5 M ethylmagnesium bromide (1.65 mmol) was added to 1.1 ml ether solvent and stirred at 0 ° C for a while. To this solution, indole (1.3 mmol) previously dissolved in 1.1 ml ether solvent was gradually added, and stirred at room temperature for 30 minutes after the addition was completed.

While stirring, 2 -naphthoyl chloride (1.46 mmol) previously dissolved in 1 ml ether solvent was gradually added. The reaction was left stirring at room temperature for 1.5 hours. Thereafter, the reaction was stopped by adding the same volume of a saturated aqueous ammonium chloride solution as the reaction solution, and stirring was continued until it became a fine powder. The powder was filtered, washed with an appropriate amount of water, and then washed with an appropriate amount of ether. The powder was dissolved in 1 ml methanol, 1 ml aqueous sodium hydroxide solution (0.4 g / ml) was added, and the mixture was stirred at room temperature for 18 hours. The precipitate was filtered and washed with an appropriate amount of methanol, water and ether. Then, it dried under vacuum at 100 ° C. to obtain 0.25 g of 3- ( 2 -naphthoyl) indole mixture. This mixture was used in the next synthetic step without purification.

After dissolving 0.2 g 3- ( 2 -naphthoyl) indole mixture with 1.5 ml dimethyl sulfoxide (DMSO), 0.6 g potassium hydroxide was added. To this reaction solution, 1-bromoheptanoic acid (5.5 mmol) was gradually added and stirred at 85 ° C. for 18 hours. The reaction solution was diluted with an appropriate amount of water and then extracted three times with an appropriate amount of ethyl acetate. The extract was concentrated and purified by chromatography (silica carrier, eluent: petroleum ether / = 7/1) to give 0.1 g 1-heptanoic acid-3- ( 2 -naphthoyl) indole (yield: 41%). I was able to get it.

  The structure of the obtained reaction product was identified as the above formula (Chemical Formula 1) by infrared spectrum and NMR data.

(2) Synthesis of immunogen 10 mg synthetic cannabinoid derivative was dissolved in 1 ml DMSO, 5 mg ethyl (dimethylaminopropyl) carbodiimide (EDC) was added, and the mixture was stirred for 1 hour at room temperature. The solution was gradually added with stirring to a solution in which 100 mg of keyhole limpet hemocyanin (KLH) was previously dissolved in a phosphate buffer solution ((pH 7.4) PBS buffer) containing 5 ml of physiological saline. Thereafter, the mixture was further stirred at room temperature for 8 hours.

  After removing the precipitate from this solution with a 0.25 μm filter in advance, the synthetic cannabinoid derivative as an immunogen is removed by removing the unreacted synthetic cannabinoid derivative with a Sephadex G-25 column (eluent: PBS buffer). Derivative-KLH (concentration: 1 mg / ml) was obtained.

(3) Immunization To the 1 mg / mL immunogen prepared above, the same volume of adjuvant (Human tuberculosis killed complete Freund's adjuvant, Wako Pure Chemicals, H37Rv) was added, and well emulsified with a homogenizer (1000 rpm). About 8 weeks old mice were injected with 10 μl of an adjuvant emulsion containing an immunogen at 10 sites.

  Two weeks later, the same volume of incomplete Freund's adjuvant was emulsified with 1 mg / mL immunogen with a homogenizer, and 100 μl of this emulsion was injected into 10 sites at a time. Complete Freund's adjuvant emulsion was similarly injected. Blood was collected one week after the injection, and the antibody production shown below was confirmed.

(4) Serum evaluation The collected serum was confirmed for antibody production by enzyme immunoassay (ELISA). Prepared with 0.1 mg / mL BSA-PBS-Az (solid solution of bovine serum albumin (hereinafter referred to as BSA) at a concentration of 0.1 mg / mL in a 0.04 wt% sodium azide PBS solution) as a solid phase 2 0.5 μg / mL synthetic cannabinoid derivative-BSA was used at 100 μl / well. Peroxidase-labeled anti-mouse IgG antibody or peroxidase-labeled anti-mouse IgM antibody was used as the second antibody. As a result, production of a synthetic cannabinoid derivative antibody was observed, and it was confirmed that an IgG / IgM ratio was 100 or more and a class switch occurred.

(5) Boosting (injection of weak immunogen) was performed in order to enlarge two spleens that had particularly high titers among the mice immunized with cell fusion. As the immunogen, a 1 mg / mL synthetic cannabinoid derivative-KLH solution was used as it was without adding an adjuvant. Spleen cells of mice that had passed 3 days after the boost were excised and fused with a mouse myeloma cell line (P3X63-Ag8.653) by a conventional method using polyethylene glycol having an average molecular weight of 1,500. Spleen cells from the same mouse were used as feeders (cells supplying growth factors), and cultured for 1 day in Ishikov medium containing 15% by weight fetal calf serum (hereinafter FCS) on two 96-well plates (100 μl). / Well) 2 × concentration of hypoxanthine / aminobuterin / thymidine (HAT) medium was added at 100 μl / well and cultured in a CO2 incubator (CO2 concentration: 5% by volume, temperature: 37 ° C., humidity: 95%) for 1 week. did. Thereafter, the culture supernatant was removed, and the culture medium was replaced with hypoxanthine / thymidine (HT) medium (250 μl / well) containing 15 wt% FCS.

(6) One week after replacement with cell-sorted HT medium, 200 μl / well of culture supernatant was taken out. HT medium (200 μl / well) containing 15% by weight of FCS was added and cultured for 3 days, after which the culture supernatant was taken out 200 μl / well. In total, 400 μl / well of the culture supernatant could be obtained. Using this culture supernatant, the binding ability to the synthetic cannabinoid derivative-BSA was measured by the ELISA method shown below. As a solid phase, 100 μl / well of a 2.5 μg / mL synthetic cannabinoid derivative-BSA solution prepared with 0.1 mg / mL BSA / PBS / Az was used. Cell culture supernatant was used as an antibody solution. As a result, there were 3 wells that showed binding ability to the synthetic cannabinoid derivative-BSA.

(7) Cloning The above 3 well cells were diluted to a concentration containing 1 cell per well (limit dilution) and dispensed into 3 96 well microplates. Early growth was stimulated using thymocytes of 5 week old mice (Balb / c) as feeders. The culture is advanced while increasing the size of the plate, and timely supernatant screening by ELISA is repeated to finally select cell lines that show high titer against synthetic cannabinoid derivative-BSA and show good growth. The culture was continued in 200 ml to a concentration of 5 × 10 5 cells / mL. Finally, three strains that showed binding ability to the synthetic cannabinoid derivative-BSA were selected.

  These cell lines were deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as Deposit No. NITE BP-01955, Deposit No. NITE BP-0195, Deposit No. NITE BP-01957. The date of deposit (original deposit date) is October 27, 2014.

(8) Storage of cells The cell line finally selected is centrifuged, and the supernatant is centrifuged at a concentration of 5 × 10 6 cells / mL to 1 mL of FCS: dimethyl sulfoxide = 9: 1 (volume ratio) solution. After floating and freezing at −80 ° C., it was transferred to −135 ° C. for long-term storage.

(9) Purification of antibody After the collected culture medium is isolated by centrifugation, the supernatant is purified from the cell culture supernatant by affinity chromatography using protein A-binding gel (Protein A Sepharose CL-4B, manufactured by Pharmacia). Monoclonal antibody was purified. This monoclonal antibody was confirmed by SDS polyacrylamide gel electrophoresis and compared with a standard protein to confirm that the purified antibody was an IgG composed of an H chain with a molecular weight of about 50,000 and an L chain with about 25,000.

(10) Evaluation of antibody The monoclonal antibody purified by the affinity chromatography was evaluated by the following enzyme immunoassay (ELISA method).

ELISA Method (A) Antigen Coating 2.5 μg / mL Synthetic Cannabinoid Derivative-BSA Combined with Synthetic Cannabinoid Derivative and Bovine Serum Albumin (BSA) -BSA Antigen on Microplate (96-well Plate Made of Vinyl Chloride) The solution was injected at 100 μl / well and stored overnight at 20 degrees. Immediately before the experiment, the antigen solution was removed with an aspirator.

(B) Blocking BSA-PBS-Az was injected at 200 μl / well and left at room temperature for 30 minutes. Then, BSA-PBS-Az was removed with an aspirator. When the subsequent experiment was not performed on the same day, the sample was stored in this state at 4 degrees with filter paper moistened with water.

(C) Antibody reaction An antibody solution diluted with 1 wt% BSA-PBS-Az (dilution ratio: 100 to 1,000,000 times) was added while shaking at 100 µl / well. After storing at room temperature for 3 hours, the antibody solution was removed with an aspirator, washed 3 times with PBS, and the remaining PBS was removed with an aspirator.

(D) Reaction of second antibody 0.2 μg / mL peroxidase-labeled anti-mouse IgG antibody derived from goat (manufactured by KPL) dissolved in 1 wt% BSA in PBS or 0.2 μg / mL peroxidase-labeled anti-antioxidant A mouse IgM antibody goat (manufactured by KPL) dissolved in 1 wt% BSA in PBS was injected at 50 μl / well and left at room temperature for 30 minutes. The sample was removed with an aspirator, washed with PBS three times, and the remaining PBS was removed with an aspirator.

(E) Reaction and termination of substrate 40 mg of O-phenylenediamine (for biochemistry) was dissolved in 10 mL of citrate monophosphate buffer (pH 5), and 4 μL of 30 wt% aqueous hydrogen peroxide was added immediately before use ( Substrate solution) was injected at 100 μl / well and allowed to stand at room temperature. After 5 minutes, the reaction was stopped by injecting 25 μl / well of 4N sulfuric acid.

(F) Measurement Absorbance at 492 nm was measured using a Toyo Soda microplate reader.

  The present invention can be used in industrial fields such as legal management and health management.

Claims (4)

An immunogen in which a compound represented by the following structural formula (Formula 1) and a carrier protein keyhole limpet hemocyanin are bound.

  A method for producing a monoclonal antibody-producing cell line, comprising the step of inoculating the immunogen according to claim 1. A monoclonal antibody-producing cell line of any one of the following, which is a cell line produced by the method for producing a monoclonal antibody-producing cell line according to claim 2 and producing a monoclonal antibody capable of binding to the compound:
A cell line deposited under deposit number NITE BP-01955,
The cell line deposited under the deposit number NITE BP-01956,
Cell line deposited under deposit number NITE BP-01957.   A monoclonal antibody produced by the cell line according to claim 3, which reacts with the compound.

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