CN118255702A - Preparation method and application of nicotine hapten and complete antigen - Google Patents
Preparation method and application of nicotine hapten and complete antigen Download PDFInfo
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- CN118255702A CN118255702A CN202410155776.4A CN202410155776A CN118255702A CN 118255702 A CN118255702 A CN 118255702A CN 202410155776 A CN202410155776 A CN 202410155776A CN 118255702 A CN118255702 A CN 118255702A
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- 229960002715 nicotine Drugs 0.000 title claims abstract description 74
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 70
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 239000000427 antigen Substances 0.000 title claims abstract description 34
- 102000036639 antigens Human genes 0.000 title claims abstract description 34
- 108091007433 antigens Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims description 20
- 238000000034 method Methods 0.000 claims abstract description 22
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000002965 ELISA Methods 0.000 claims abstract description 9
- TZRQZPMQUXEZMC-UHFFFAOYSA-N tert-butyl n-(2-bromoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCBr TZRQZPMQUXEZMC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000003571 electronic cigarette Substances 0.000 claims abstract description 5
- BMIBJCFFZPYJHF-UHFFFAOYSA-N 2-methoxy-5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine Chemical compound COC1=NC=C(C)C=C1B1OC(C)(C)C(C)(C)O1 BMIBJCFFZPYJHF-UHFFFAOYSA-N 0.000 claims abstract description 4
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- 238000012360 testing method Methods 0.000 claims abstract description 3
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- 150000001875 compounds Chemical class 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 24
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- 238000006243 chemical reaction Methods 0.000 claims description 16
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 16
- 238000001228 spectrum Methods 0.000 claims description 15
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 235000019502 Orange oil Nutrition 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
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- KTYAQHYBYRVCGD-UHFFFAOYSA-N [Ir].COC1=CC=CCCCC1 Chemical class [Ir].COC1=CC=CCCCC1 KTYAQHYBYRVCGD-UHFFFAOYSA-N 0.000 claims description 5
- 230000003053 immunization Effects 0.000 claims description 5
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 3
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 3
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 3
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 3
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000007872 degassing Methods 0.000 claims description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims description 3
- 108060003552 hemocyanin Proteins 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 238000002649 immunization Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims description 3
- 235000019198 oils Nutrition 0.000 claims description 3
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- 229940034208 thyroxine Drugs 0.000 claims description 3
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims description 3
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- 230000015572 biosynthetic process Effects 0.000 claims description 2
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims 8
- 239000007864 aqueous solution Substances 0.000 claims 1
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- LSJOYRIJHIYLLP-UHFFFAOYSA-N CO[Ir] Chemical class CO[Ir] LSJOYRIJHIYLLP-UHFFFAOYSA-N 0.000 abstract 1
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
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- UIKROCXWUNQSPJ-VIFPVBQESA-N (-)-cotinine Chemical compound C1CC(=O)N(C)[C@@H]1C1=CC=CN=C1 UIKROCXWUNQSPJ-VIFPVBQESA-N 0.000 description 1
- MYKUKUCHPMASKF-VIFPVBQESA-N (S)-nornicotine Chemical compound C1CCN[C@@H]1C1=CC=CN=C1 MYKUKUCHPMASKF-VIFPVBQESA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- UIKROCXWUNQSPJ-UHFFFAOYSA-N Cotinine Natural products C1CC(=O)N(C)C1C1=CC=CN=C1 UIKROCXWUNQSPJ-UHFFFAOYSA-N 0.000 description 1
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- MYKUKUCHPMASKF-UHFFFAOYSA-N Nornicotine Natural products C1CCNC1C1=CC=CN=C1 MYKUKUCHPMASKF-UHFFFAOYSA-N 0.000 description 1
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Abstract
A process for preparing nicotine hapten and complete antigen includes such steps as preparing 5-tetraalkoxyethylboryl-nicotine by the boration reaction of nicotine and bisboronic acid pinacol ester under the catalysis of methoxy iridium dimer, preparing 5-hydroxy-nicotine by introducing hydrogen peroxide, adding tert-butyl (2-bromoethyl) carbamate, introducing tert-butyl (2-bromoethyl) carbamate to 5-hydroxy-nicotine, and adding trifluoroacetic acid. The antigen of the invention presents specific nicotine antigenic determinant, and the produced antibody has high specificity and high sensitivity, has no obvious cross reaction on nicotine metabolite, and can be used for establishing an enzyme-linked immunoassay method, a colloidal gold test paper method and the like, thereby realizing the rapid detection of nicotine in electronic cigarettes, heating non-combustion products and smoke-like products.
Description
Technical Field
The invention relates to a preparation method and application of nicotine hapten and complete antigen. Belongs to the technical field of biochemical engineering.
Background
Electronic cigarette/cigarette-like product management is an innovative and challenging task with great difficulty. With the rapid development of Internet economy, micro-communication and other self-media and live broadcast software such as sound shaking and fast manual broadcasting are rapidly popularized, and some merchants rapidly realize advertisement release, information exchange, fund transaction and physical circulation of the smoke-like products by means of a network platform and a logistics delivery enterprise, so that the supervision of the smoke-like products needs to have higher timeliness, and classification and identification can be rapidly carried out on the smoke-like products on sales and circulation sites. The most important means of this is the screening assay for nicotine in such products. The current nicotine determination methods are almost established based on photometric analysis or chromatographic techniques, and the methods have the characteristics of accurate results and high reliability, but have long analysis period and high technical requirements and can only be completed in professional laboratories. The lack of on-site and rapid detection technology of nicotine in the smoke-like products is a bottleneck for comprehensively and effectively supervising the smoke-like products in the industry.
Immunoassay is a very efficient, sensitive and rapid detection method, however, obtaining monoclonal antibodies with high affinity and specificity is a precondition for immunological detection, and the synthesis of artificial antigens is an important step.
Disclosure of Invention
The invention aims to provide a preparation method and application of nicotine hapten and complete antigen, and the prepared product can be used for rapidly detecting electronic cigarettes/cigarette-like products containing nicotine.
The invention aims at realizing the following technical scheme:
The nicotine hapten adopted by the invention is preferably selected from 8 nicotine haptens, the screening principle is based on molecular rigid structure and immunological experience, particularly, hapten structures of different substituent groups on a nicotine No. 5 position are compared with animal immune serum data, meanwhile, serum data of carboxyl hapten and amino hapten are examined, and experimental results show that the amino hapten at the No. 5 position has a good immunological result, wherein the amino hapten-5 (namely the nicotine hapten) is the preferred hapten structure.
The preparation method of the nicotine hapten comprises the steps of carrying out a boration reaction on nicotine and bisboronic acid pinacol ester under the catalysis of methoxy (cyclooctadiene) iridium dimer to prepare 5-tetraalkoxy ethylboron-nicotine (compound 3), introducing hydrogen peroxide to prepare 5-hydroxy-nicotine (compound 4), introducing tert-butyl (2-bromoethyl) carbamate on the compound 4, and finally adding trifluoroacetic acid to prepare the nicotine hapten 5-amino-ethoxy-nicotine, wherein the molecular structural formula is as follows:
the specific steps are as follows (see synthetic route shown in fig. 1):
1) Dipinacol biborate (2.253 g), 4 '-di-tert-butyl-2, 2' -bipyridine (164.2 mg), methoxy (cyclooctadiene) iridium dimer (198.4 mg) and nicotine (1.6 mL,10 mmol) were dissolved in a sealed container. The vessel was evacuated and refilled with nitrogen three times by freeze-pump-thaw degassing. The reaction mixture was heated in the sealed vessel at 110 ℃ overnight. The reaction mixture was cooled to room temperature and the solvent was evaporated to give compound 3 (orange oil, 90%, detected by GC-MS).
2) To a stirred solution of compound 3 (about 9 mmol) in acetic acid (18 mL, 0.5M) was added dropwise hydrogen peroxide (1 mL,1.0 eq, 30% in water) at 0deg.C. After 30min, the reaction mixture was warmed to room temperature until complete conversion (monitored by TLC) for about 4 hours. Evaporation of the reaction mixture gave compound 4 (orange oil), which was purified by chromatography (ethyl acetate/methanol=3:1) in 75% yield (1.344 g).
3) Compound 4 (1.344 g) and cesium carbonate (3.61 g) were stirred in CH 3 CN (38 ml,0.2 m) at room temperature for 30min. Tert-butyl (2-bromoethyl) carbamate 5 (2.00 g) was added and the reaction mixture was stirred overnight at 80℃in air. After cooling to ambient temperature, the reaction mixture was evaporated to give compound 6 (yellow oil), which was purified by chromatography (ethyl acetate/petroleum ether=9/1) in 60% (1.465 g). And simultaneously, nuclear magnetic characterization (H spectrum and C spectrum) is carried out on the compound 6, which shows that the chemical synthesis structure and purity reach the expectations.
4) To a solution of compound 6 (1.465 g) in CH 2Cl2 (9 ml,0.5 m) was added TFA (5 ml, vtfa/VDCM =1:5) at 0 ℃. The solution was stirred for 12 hours after warming to room temperature. The mixture was evaporated under vacuum and then purified by chromatography (ethyl acetate/methanol=4/1) to give an amino hapten-5 yield of 60% (568 mg); and simultaneously, nuclear magnetic characterization (H spectrum and C spectrum) is carried out on the amino hapten-5, which shows that the chemical synthesis structure and purity reach the expectations.
The nicotine hapten can be used for preparing an antigen system raw material for animal immunization.
The nicotine hapten is coupled with carrier protein, which is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin and thyroxine, and the carrier protein is prepared by the preparation method.
The preparation method comprises the following specific steps:
Preparation of immune antigen: amino hapten-5. Mu.L was added to 430. Mu.L DMSO, DSG 10.33mg was added, and stirred at room temperature for 4h. 20mg of BSA was dissolved in 2.8mL of 50mM boric acid buffer solution having pH of 8.5, and after complete dissolution, 0.7mL of DMSO was added and allowed to stand at 4 ℃. The hapten solution was added to the BSA solution 10 times at 10min intervals and allowed to react overnight with stirring at 4 ℃. 10. Mu.L of ethanolamine was added thereto and stirred at room temperature for 1 hour. Desalting by desalting column, adding NaN 3, and storing at-20deg.C.
Preparation of the coating antigen: amino hapten-5. Mu.L was added to 430. Mu.L DMSO, DSC 8.11mg was added and stirred at room temperature for 4h. To 20mg of OVA, 2.8mL of 50mM boric acid buffer solution having a pH of 8.5 was added 0.7mL of DMSO after complete dissolution, and the mixture was allowed to stand at 4 ℃. The hapten solution was added to the BSA solution 10 times at 10min intervals and allowed to react overnight with stirring at 4 ℃. 10. Mu.L of ethanolamine was added thereto and stirred at room temperature for 1 hour. Desalting by desalting column, adding NaN 3, and storing at-20deg.C.
The monoclonal antibody obtained by immunizing animals with the complete antigen prepared by the invention has high specificity and high sensitivity, has no obvious cross reaction on nicotine metabolites, and can be used for establishing an enzyme-linked immunoassay method and a colloidal gold and fluorescence rapid detection test paper method, thereby realizing rapid detection of nicotine in electronic cigarettes, heating non-combustion products and smoke-like products.
Drawings
Fig. 1: a synthetic route pattern of nicotine hapten;
fig. 2: compound 3 reaction and reaction tracking;
fig. 3: compound 4 reaction and reaction tracking;
fig. 4: compound 6 reaction and reaction tracking;
fig. 5: compound 7 reaction and reaction tracking;
Fig. 6: compound 6 nuclear magnetic resonance hydrogen spectrum;
fig. 7: compound 6 nuclear magnetic resonance carbon spectrum;
Fig. 8: compound 7 nuclear magnetic resonance hydrogen spectrum;
Fig. 9: compound 7 nuclear magnetic resonance carbon spectrum;
Fig. 10: ultraviolet absorption spectrum of nicotine complete antigen.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention. In addition, various changes or modifications may be made by those skilled in the art within the scope of the appended claims, and such changes or modifications should also fall within the scope of the invention.
EXAMPLE 1 preparation of nicotine hapten
1. Preparation of nicotine hapten
1) Dipinacol biborate (2.253 g), 4 '-di-tert-butyl-2, 2' -bipyridine (164.2 mg), methoxy (cyclooctadiene) iridium dimer (198.4 mg) and nicotine (1.6 mL,10 mmol) were dissolved in a sealed container. The vessel was evacuated and refilled with nitrogen three times by freeze-pump-thaw degassing. The reaction mixture was heated in the sealed vessel at 110 ℃ overnight. The reaction mixture was cooled to room temperature and the solvent was evaporated to give compound 3 (orange oil, 90%, detected by GC-MS).
2) To a stirred solution of compound 3 (about 9 mmol) in acetic acid (18 mL, 0.5M) was added dropwise hydrogen peroxide (1 mL,1.0 eq, 30% in water) at 0deg.C. After 30min, the reaction mixture was warmed to room temperature until complete conversion (monitored by TLC) for about 4 hours. Evaporation of the reaction mixture gave compound 4 (orange oil), which was purified by chromatography (ethyl acetate/methanol=3:1) in 75% yield (1.344 g).
3) Compound 4 (1.344 g) and cesium carbonate (3.61 g) were stirred in CH 3 CN (38 ml,0.2 m) at room temperature for 30min. Tert-butyl (2-bromoethyl) carbamate 5 (2.00 g) was added and the reaction mixture was stirred overnight at 80℃in air. After cooling to ambient temperature, the reaction mixture was evaporated to give compound 6 (yellow oil), which was purified by chromatography (ethyl acetate/petroleum ether=9/1) in 60% (1.465 g). And simultaneously, nuclear magnetic characterization (H spectrum and C spectrum) is carried out on the compound 6, which shows that the chemical synthesis structure and purity reach the expectations.
4) To a solution of compound 6 (1.465 g) in CH 2Cl2 (9 ml,0.5 m) was added TFA (5 ml, vtfa/VDCM =1:5) at 0 ℃. The solution was stirred for 12 hours after warming to room temperature. The mixture was evaporated under vacuum and then purified by chromatography (ethyl acetate/methanol=4/1) to give the amino hapten-5 in a yield of 60% (568 mg). And simultaneously, nuclear magnetic characterization (H spectrum and C spectrum) is carried out on the amino hapten-5, which shows that the chemical synthesis structure and purity reach the expectations.
2. Identification of nicotine hapten
The reaction trace of the intermediate and nicotine hapten is shown in FIGS. 2-9 for 1H NMR,13 C NMR.
EXAMPLE 2 preparation of nicotine complete antigen
The nicotine complete antigen is obtained by coupling nicotine hapten and carrier protein, wherein the carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin and thyroxine.
The present invention uses two schemes, DSG and DSC, to covalently couple nicotine hapten to macromolecules, exemplified by proteins BSA (bovine serum albumin) and OVA (ovalbumin), the specific coupling designs are shown below.
1. Preparation of immune antigens
Coupling nicotine hapten and bovine serum albumin to obtain the immune antigen.
Amino hapten-5. Mu.L was added to 430. Mu.L DMSO, DSG 10.33mg was added, and stirred at room temperature for 4h. 20mg of BSA was dissolved in 2.8mL of 50mM boric acid buffer solution having pH of 8.5, and after complete dissolution, 0.7mL of DMSO was added and allowed to stand at 4 ℃. The hapten solution was added to the BSA solution 10 times at 10min intervals and allowed to react overnight with stirring at 4 ℃. 10. Mu.L of ethanolamine was added thereto and stirred at room temperature for 1 hour. Desalting by desalting column, adding NaN 3, and storing at-20deg.C.
2. Preparation of coated antigen
Coupling nicotine hapten and ovalbumin to obtain the coating antigen.
Amino hapten-5. Mu.L was added to 430. Mu.L DMSO, DSC 8.11mg was added and stirred at room temperature for 4h. To 20mg of OVA, 2.8mL of 50mM boric acid buffer solution having a pH of 8.5 was added 0.7mL of DMSO after complete dissolution, and the mixture was allowed to stand at 4 ℃. The hapten solution was added to the BSA solution 10 times at 10min intervals and allowed to react overnight with stirring at 4 ℃. 10. Mu.L of ethanolamine was added thereto and stirred at room temperature for 1 hour. Desalting by desalting column, adding NaN 3, and storing at-20deg.C.
2. Identification of nicotine complete antigen
The complete antigen adopts ultraviolet spectrum method to identify the coupling result, and the coupling ratio is calculated by using the concentration of small molecule and protein in the conjugate. The maximum absorption peak of nicotine hapten-carrier protein is obviously changed compared with that of nicotine hapten and carrier protein, which indicates that the preparation of nicotine-carrier protein is successful.
EXAMPLE 3 preparation of Nicotine monoclonal antibodies
1. Immunization of animals
Balb/c female mice of 6-8 weeks old are selected, immune antigens are mixed and emulsified with Freund's adjuvant, and the mice produce antisera according to 100 mug/dose of immunity.
2. Cell fusion and subcloning
Taking the Balb/c mouse spleen cells which are successfully immunized, adjusting the ratio of the myeloma cells SP2/0 to the immunized mouse spleen cells to be 1:5-1:10 for fusion, measuring cell culture supernatant by adopting a competition ELISA method, and screening out proper positive holes. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
3. Preparation and purification of monoclonal antibodies
Preparing ascites: balb/c female mice with the age of 10-11 weeks are selected, the abdominal cavity of the Balb/c mice is injected with an adjuvant, each 0.3-0.5 mL of the abdominal cavity is treated for about 7-15 days, and the screened hybridoma cell strains are inoculated into the abdominal cavity of the Balb/c mice after the expanded culture. Mice were sacrificed by cervical spine removal and were ascites, centrifuged at 3000rpm for 10min.
Purification of monoclonal antibodies: and (3) firstly purifying the ascites by an ammonium sulfate precipitation method, and then purifying by a Protein G column to obtain the final monoclonal antibody.
4. Determination of the titers of monoclonal antibodies
The titer of the antibody was determined by the Elisa method to be 1:600000.
Competitive Elisa, namely, coating an ELISA plate by nicotine-OVA, adding a nicotine standard substance and a nicotine monoclonal antibody, incubating for 2 hours at 37 ℃, washing, adding an HRP-marked goat anti-mouse secondary antibody, washing again, adding TMB color development liquid, stopping the reaction by acid, and measuring the absorbance value at 450nm on an ELISA.
4. Monoclonal antibody specificity assay
The specificity of an antibody refers to the ability to recognize a substance with a corresponding antigen or near antigen. The antibody has high specificity and strong recognition capability. The measured specificity is usually expressed in terms of cross-reactivity. The cross-reactivity can be determined using a competitive inhibition assay. The competition inhibition curves were made with different concentrations of antigen and similar antigen, the respective binding rates were calculated, the concentrations at the IC50 were determined, and the cross-reaction rates were calculated according to the following formula.
The present invention tested the cross-reactivity of antibodies to nicotine metabolites and analogues calculated according to the above formula: the Equisetum is less than 1%, cotinine is less than 1%, neonicotinoid is less than 1%, nornicotine is less than 1%, mastin is less than 1%, and beta-dien nicotine is less than 1%, which indicates that the antibody specificity is better.
Example 4 preparation of enzyme-Linked immunosorbent assay kit for detecting nicotine content
Preparing a nicotine content detection enzyme-linked immunosorbent assay kit, which comprises the following components:
1) The ELISA plate is coated with antigen;
Preparing an ELISA plate: diluting the coating antigen to 0.1 mu g/mL with a coating buffer solution, adding 100 mu L of the coating buffer solution into each hole, coating at 4 ℃ overnight, spin-drying the liquid, washing 3 times with 0.05% of PBST, beating to dryness, adding 200 mu L of 5% of skimmed milk powder into each hole to seal an ELISA plate, sealing for 2 hours at 37 ℃, spin-drying the liquid, washing 3 times with 0.05% of PBST, beating to dryness, drying in an oven completely, and vacuumizing with an aluminum foil bag for preservation.
2) Nicotine standard solution: the concentration is 0, 0.01 mug/mL, 0.05 mug/mL, 0.25 mug/mL, 1 mug/mL, 5 mug/mL, 20 mug/mL in sequence;
3) Nicotine antibody working fluid;
4) HRP-labeled goat anti-mouse secondary antibody;
5) TMB color development liquid;
6) Stop solution: 2M H 2SO4 or 4M HCl;
7) Washing liquid: 0.05% pbst;
8) And (3) a complex solution: 10mM phosphate buffer, pH 7.4.
Example 5 detection of nicotine content in tobacco products
1. Sample pretreatment
Accurately weighing 0.1g of the sample, adding 10mL of 0.5% PBST solution into a 15mL centrifuge tube, vibrating and extracting for 30min on a vibrator, standing, taking supernatant, and diluting 100 times by adopting the 0.5% PBST solution.
2. Kit detection
Adding 50 mu L of standard substance/sample into corresponding microplate reader micropores, adding 50 mu L/hole of antibody working solution, shaking, mixing, incubating at 37 ℃ for 1h, spin-drying liquid, washing for 3 times according to 200 mu L/hole washing liquid, and spin-drying liquid. Adding HRP-labeled goat anti-mouse secondary antibody 100 mu L/hole, shaking and mixing well, incubating at 37 ℃ for 0.5h, and taking out the repeated washing plate. Adding 100 mu L/hole of the color development liquid, incubating for 15min at 37 ℃, and shaking and mixing uniformly. 50. Mu.L/Kong Zhongzhi of liquid was added. The microplate reader was set at 450nm, the reference wavelength was 620nm, and the OD value per well was determined.
3. Analysis of detection results of kit
The percent absorbance of a nicotine standard or sample is equal to the average of the absorbance value of the standard or sample divided by the absorbance value of the first standard, and multiplied by 100 to obtain the percent absorbance value of the standard or sample. And drawing a standard curve graph by taking the percentage absorbance of the standard substance as an ordinate and the logarithm of the concentration of the nicotine standard substance as an abscissa. Substituting the percent absorbance of the sample into a standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the dilution multiple corresponding to the standard curve to obtain the actual concentration of nicotine in the sample.
Claims (8)
1. A preparation method of nicotine hapten is characterized by comprising the following steps: the 5-tetraethoxyboron-nicotine (compound 3) is prepared by the boration reaction of nicotine and bisboronic acid pinacol ester under the catalysis of methoxy (cyclooctadiene) iridium dimer, hydrogen peroxide is introduced to prepare 5-hydroxy-nicotine (compound 4), tert-butyl (2-bromoethyl) carbamate is introduced to the compound 4, and trifluoroacetic acid (Trifluoroacetic acid, TFA) and dichloromethane (Dichloromethane, DCM) are finally added to prepare the nicotine hapten 5-amino-ethoxy-nicotine (amino hapten-5), wherein the molecular structural formula is as follows:
The specific reaction process, namely the synthesis route, is as follows:
2. The method of preparing a nicotine hapten according to claim 1, wherein: the method comprises the following specific steps:
1) Nicotine (1.6 ml,10 mmol), pinacol ester of bisborate (2.253 g), 4 '-di-tert-butyl-2, 2' -bipyridine (164.2 mg) and methoxy (cyclooctadiene) iridium dimer (198.4 mg) and dissolved in a sealed vessel that was evacuated and refilled with nitrogen three times by freeze-pump-thaw degassing, the reaction mixture was heated overnight in the sealed vessel at 110 ℃, the reaction mixture was cooled to room temperature, and the solvent was evaporated to give compound 3 (orange oil, 90%, detected by GC-MS);
2) To a stirred solution of compound 3 (about 9 mmol) in acetic acid (18 ml,0.5 m) was added dropwise hydrogen peroxide (1 ml,1.0 eq, 30% aqueous solution) at 0 ℃ for 30min, after which the reaction mixture was warmed to room temperature until complete conversion (TLC monitoring) for about 4 hours, the reaction mixture was evaporated to give compound 4 (orange oil), which was purified by chromatography (ethyl acetate/methanol=3:1) in 75% (1.344 g);
3) Compound 4 (1.344 g) and cesium carbonate (3.61 g) were stirred in CH 3 CN (38 ml,0.2 m) at room temperature for 30 min, tert-butyl (2-bromoethyl) carbamate 5 (2.00 g) was added, then the reaction mixture was stirred in air at 80 ℃ overnight, after cooling to ambient temperature, the reaction mixture was evaporated to give compound 6 (yellow oil), which was purified by chromatography (ethyl acetate/petroleum ether=9/1), yield 60% (1.465 g) while subjecting compound 6 to nuclear magnetic characterization (H-spectrum and C-spectrum) indicating that the chemical synthesis structure and purity were expected;
4) To a solution of compound 6 (1.465 g) in CH 2Cl2 (9 ml,0.5 m) was added TFA (5 ml, vtfa/VDCM =1:5) at 0 ℃, the solution was stirred for 12 hours after warming to room temperature, the mixture was evaporated under vacuum and then purified by chromatography (ethyl acetate/methanol=4/1) to give a 60% yield of amino hapten-5 (568 mg) while nuclear magnetic characterization (H and C spectra) was performed on amino hapten-5, indicating that the chemical synthesis structure and purity were expected.
3. Use of a nicotine hapten prepared by the method of claim 1 or 2, characterized in that: the nicotine hapten can be used for preparing an antigen system raw material for animal immunization.
4. A preparation method of nicotine complete antigen is characterized in that: is obtained by coupling nicotine hapten prepared according to claim 1 or 2 with carrier protein.
5. The method for preparing nicotine complete antigen of claim 4, wherein the carrier protein is hemocyanin, bovine serum albumin, ovalbumin, human serum albumin, thyroxine.
6. The method for preparing the nicotine complete antigen of claim 4 or 5, wherein the method comprises the following steps: the method comprises the following specific steps: adding 5 mu L of amino hapten into 430 mu L of DMSO, adding 10.33mg of DSG, and stirring for 4 hours at room temperature; 20mg of BSA was dissolved in 2.8mL of 50mM boric acid buffer solution having a pH of 8.5, and after complete dissolution, 0.7mL of DMSO was added and allowed to stand at 4 ℃; adding hapten solution into BSA solution for 10 times, and stirring overnight at 4 ℃ for reaction at intervals of 10min each time; adding 10 mu L of ethanolamine, and stirring for 1h at room temperature; desalting by desalting column, adding NaN 3, and storing at-20deg.C.
7. The method for preparing the nicotine complete antigen of claim 4 or 5, wherein the method comprises the following steps: the method comprises the following specific steps: amino hapten-5 μl was added to 430 μl DMSO, DSC 8.11mg was added, and stirred at room temperature for 4h; 20mg of OVA is dissolved in 2.8mL of 50mM boric acid buffer solution with pH of 8.5, after the dissolution is completed, 0.7mL of DMSO is added, and the mixture is left to stand at 4 ℃; adding the hapten solution into the BSA solution for 10 times, stirring overnight at 4 ℃ for reaction at intervals of 10 min; adding 10 mu L of ethanolamine, and stirring for 1h at room temperature; desalting by desalting column, adding NaN 3, and storing at-20deg.C.
8. Use of a nicotine complete antigen prepared by the method of claims 4-7, characterized in that: the monoclonal antibody obtained by immunizing animals with the nicotine complete antigen has high specificity and high sensitivity, has no obvious cross reaction on nicotine metabolites, and can be used for establishing an enzyme-linked immunoassay method and a colloidal gold and fluorescence rapid detection test paper method, thereby realizing rapid detection of nicotine in electronic cigarettes, heating non-combustion products and smoke-like products.
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| CN118255702A true CN118255702A (en) | 2024-06-28 |
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