2-methylisoborneol hapten, its preparation method and application

Abstract

The application relates to the technical field of 2-methyl isochroman alcohol detection, in particular to a 2-methyl isochroman alcohol hapten, a preparation method and application thereof. The 2-methyl isochroman alcohol hapten has a structural formula as shown in formula (I). The application uses D-camphor as raw material, and a novel 2-methyl isochroman alcohol hapten compound shown in formula (I) is prepared, specific antibodies prepared by the method are used for detecting 2-methyl isochroman alcohol, have excellent specific recognition, and the IC 50 can reach 0.46 ng / mL.

Description

Technical Field

[0001] This invention relates to the field of 2-methylisoborneol detection technology, specifically to a 2-methylisoborneol hapten, its preparation method, and its application. Background Technology

[0002] In recent years, with the increasing eutrophication of surface waters such as rivers and lakes, drinking water quality problems caused by odorous substances secreted by the excessive growth of algae and actinomycetes have become a research hotspot in the fields of drinking water, aquaculture, and the environment. Among them, 2-methylisoborneol (2-MIB) is one of the important pollutants causing earthy odors in drinking water and aquaculture.

[0003] Currently, the detection methods for 2-MIB in water and aquatic products, both domestically and internationally, mainly rely on gas chromatography and gas chromatography-mass spectrometry, supplemented by pretreatment techniques such as purge and trap, headspace solid-phase microextraction, and dual-flow headspace thermal desorption. These instruments have high analytical sensitivity, but they require expensive gas chromatography and mass spectrometry equipment and professional technicians, which limits their application scope.

[0004] CN116754622A relates to a method for detecting 2-methylisoborneol in water. It involves synthesizing a novel two-dimensional MOF using ferrocene dicarboxylic acid and ferrocene carboxylic acid as organic ligands and copper ions as metal sites. The two-dimensional MOF is then composited with edge-alkenylated MXene to obtain alkenylated MXene@defective sheet-like CuFc-MOFs. A molecularly imprinted polymer is then selectively polymerized onto the edges of the alkenylated MXene to obtain alkenylated MXene@defective sheet-like CuFc-MOFs / MIP. An alkenylated MXene@defective sheet-like CuFc-MOFs / MIP modified electrode is then prepared using a drop-coating method. This modified electrode is placed in a phosphate buffer solution containing potassium chloride and potassium ferricyanide, and electrochemical sensing detection of 2-MIB in water is achieved using differential pulse voltammetry. This patented method achieves a limit of detection (LOD) of 30 pg / L and exhibits a wide linear range. However, the preparation process of the modified electrode is complex and has low reproducibility.

[0005] Immunoassay methods (such as ELISA and immunochromatographic strips) are assays based on the binding between antigens and antibodies. While maintaining detection performance, they also enable on-site, real-time testing, significantly reducing the skill requirements for specialized operators. The specific binding of antigen and antibody is crucial for detection sensitivity and accuracy; therefore, the structural design of the hapten is particularly important for obtaining high-performance antigens and antibodies. Thus, developing highly specific 2-methylisoborneol hapten / artificial antigens is of paramount importance for a rapid, highly sensitive, and low-cost detection method for 2-methylisoborneol.

[0006] Professor Deng Anping's research group at Soochow University reported a 2-methylisoborneol hapten compound with the following structural formula:

[0007] However, the results of the antiserum detection test after mouse immunization showed that the inhibition rate of 3 μg / mL 2-methylisoborneol against the most sensitive antiserum was only 60.6%, which was poor and could not meet the needs of developing a rapid immunoassay method for 2-methylisoborneol. In addition, this study did not ultimately obtain a usable monoclonal antibody (Liu Chang. Preparation of monoclonal antibodies of chloramphenicol and dimethylisoborneol and establishment of enzyme-linked immunosorbent assay method [D]. Soochow University.).

[0008] Patent CN 112358549 A discloses the hapten structure of 2-methylisoborneol (as reported by Professor Deng Anping's research group). Furthermore, mouse monoclonal antibodies were prepared, and based on this, an indirect competitive ELISA method for detecting 2-MIB was established. 50 The value was 52.61 ng / mL, which again proves that the sensitivity of the antibody prepared by this hapten structure is insufficient and cannot meet the needs of the development of a rapid immunoassay method for 2-MIB.

[0009] A journal article (Plhak LC, Park ES. High-affinity monoclonal antibodies for detection of the microbial metabolite, 2-methylisoborneol. J Agric Food Chem. 2003 Jun 18;51(13):3731-6.) also reported a 2-MIB hapten structure, the structural formula of which is as follows:

[0010]

[0011] However, the sensitivity (IC50 value of 19 μg / L) of the monoclonal antibody prepared from this hapten still needs to be further improved.

[0012] Furthermore, the specificity of the antibodies obtained from the aforementioned hapten for 2-methylisoborneol is not good enough. For example, the structural similarity to structural analogs such as isoborneol, camphor, camphorquinone, and geosmin is ≥90%, or even ≥95%. This results in a high cross-reactivity rate (CR) for these structurally similar substances, even exceeding 30%. This indicates that the selectivity of the 2-methylisoborneol hapten in the existing technology needs to be improved. Summary of the Invention

[0013] This invention aims to address the problems of existing 2-methylisoborneol (2-MIB) detection methods, which rely on large instruments, are complex to operate, and are difficult to implement rapidly on-site. This invention provides a 2-methylisoborneol hapten compound, an artificial antigen, and its antibody. This antibody exhibits high specificity and affinity, and the indirect competitive ELISA analysis method based on this antibody has high sensitivity.

[0014] The first objective of this invention is to provide a 2-methylisoborneol hapten with the structural formula shown in formula (Ⅰ);

[0015] Equation (I)

[0016] This invention uses D-camphor as a starting material, which is inexpensive. An alkenyl group is introduced via a Grignard reaction, followed by reaction with mercaptoacetic acid to obtain the 2-methylisoborneol hapten compound shown in formula (I). The raw materials are inexpensive and readily available, the reaction conditions are mild, and the process is environmentally friendly, making it suitable for industrial production.

[0017] A second objective of this invention is to provide a method for preparing the 2-methylisoborneol hapten shown in formula (I), the synthetic route of which is as follows:

[0018]

[0019] Furthermore, the method for preparing the 2-methylisoborneol hapten shown in formula (I) includes the following steps:

[0020] (S1) Under an inert atmosphere, D-camphor solution (CAS: 464-49-3) was added to allyl magnesium bromide solution and reacted at -30℃ to -10℃. After the reaction was complete, the Grignard reaction in step (S1) was quenched, and the product was extracted, dried, filtered, concentrated, and purified to obtain intermediate (IA).

[0021] (S2) Under an inert atmosphere, intermediate (IA) reacts with mercaptoacetic acid to give product compound (I).

[0022] Furthermore, the inert atmosphere is nitrogen and / or argon.

[0023] Further, in step (S1), the solvent for the D-camphor solution is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, ethanol, ethyl acetate, and butyl acetate; the concentration of the D-camphor solution is 0.5-1.5 mol / L, preferably 0.8-1 mol / L; the solvent for the allyl magnesium bromide solution is selected from at least one of diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, and dichloromethane; the concentration of the allyl magnesium bromide solution is 0.5-1.5 mol / L, preferably 0.8-1 mol / L.

[0024] Further, in step (S1), the molar ratio of D-camphor to allyl magnesium bromide is 1:1-3, preferably 1:1.5-2.

[0025] Further, in step (S1), the reaction time is 4-10 h, the quenching reaction is carried out by adding pure water, the extraction is carried out by extracting with ethyl acetate, and the drying is carried out by drying with a desiccant selected from at least one of anhydrous sodium sulfate and anhydrous magnesium sulfate; the concentration is carried out by vacuum concentration to obtain an oily substance; the separation and purification are carried out by silica gel chromatography column, and the eluent is petroleum ether:ethyl acetate at a volume ratio of 3-5:1.

[0026] Further, in step (S2), the molar ratio of intermediate (IA) to mercaptoacetic acid is 1-1.5:1-1.5, preferably 1-1.1:1-1.1. The reaction between the alkenyl and mercapto groups is rapid and complete, and the reactants are fed at a molar ratio close to 1:1. After the reaction in step (S2) is complete, an oily substance is obtained; separation and purification are carried out by silica gel chromatography, with petroleum ether and ethyl acetate as the eluent at a volume ratio of 3-5:1.

[0027] A third objective of this invention is to provide a 2-methylisoborneol artificial antigen, which is obtained by conjugating the 2-methylisoborneol hapten of formula (I) with a carrier protein. The carrier protein is selected from at least one of keyhole hemocyanin, bovine serum albumin, hemocyanin, thyroprotein, and ovalbumin. Considering factors such as the success rate of antigen preparation, immunogenicity, and stability, keyhole hemocyanin (KLH) is preferably used as the carrier protein. The conjugation method is either the active ester method or the mixed anhydride method.

[0028] A fourth objective of the present invention is to provide a method for preparing the above-mentioned 2-methylisoborneol artificial antigen, comprising the following steps: coupling a carrier protein to the carboxyl group of the 2-methylisoborneol hapten shown in formula (I) by carbodiimide method.

[0029] Specifically, the preparation method of the 2-methylisoborneol artificial antigen includes the following steps:

[0030] (1) The artificial hapten shown in formula (Ⅰ) is dissolved in DMF to obtain a hapten solution;

[0031] (2) Take the hapten solution, add the carboxyl activator, and react;

[0032] (3) The carrier protein solution was dissolved in PBS buffer to obtain the carrier protein solution;

[0033] (4) Add the solution from step (2) that has completed the carboxyl activation reaction to the protein solution prepared in step (3);

[0034] (5) Dialyze the liquid phase obtained in step (4) in a dialysis bag with a molecular weight cutoff of 7-10 KDa at 2-8°C for 72-96 h, and change the buffer every 12-14 h.

[0035] Further, in step (1), the concentration of the artificial hapten shown in formula (I) is 15-20 mg / mL; in step (2), the carboxyl activator is a combination of EDC and NHS (molar ratio of 1-2:1-2); the molar ratio of the carboxyl activator to the artificial hapten shown in formula (I) is 1.2-1.5:1; step (2) is carried out at room temperature and stirred at 200-300 rpm for 2-3 h; in step (3), the volume-to-mass ratio of PBS buffer to carrier protein is 3-4 mL: 45-75 mg; in step (4), the mass ratio of formula (I) in the reaction solution of step (2) to KLH in the solution of step (3) is 1.5-2.5:6-8.

[0036] A fifth object of the present invention is to provide a 2-methylisoborneol antibody, wherein the 2-methylisoborneol antibody is obtained by immunizing animals with the above-mentioned 2-methylisoborneol artificial antigen. Further, the antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody.

[0037] A sixth object of the present invention is to provide any of the following uses for the 2-methylisoborneol hapten shown in formula (I) above, or the 2-methylisoborneol artificial antigen above, or the 2-methylisoborneol antibody above:

[0038] (1) Immunoassay methods / products for the detection of 2-methylisoborneol;

[0039] (2) Used for the detection of 2-methylisoborneol;

[0040] The analytical methods / product types include, but are not limited to, immunochromatography, membrane strips, reagent kits, chips, models, or detection platforms.

[0041] In some embodiments of the present invention, the analytical method is an indirect competitive enzyme-linked immunosorbent assay (ELISA).

[0042] The present invention has achieved the following beneficial effects:

[0043] This invention uses inexpensive and readily available D-camphor as a raw material to prepare a novel 2-methylisoborneol hapten compound of formula (I). The specific antibody prepared from this compound exhibits excellent specific recognition for the detection of 2-methylisoborneol, and can be used to establish an IC50-weighted indirect competitive ELISA. 50 It can reach 0.46 ng / mL. Attached Figure Description

[0044] Figure 1 This is the mass spectrum of the 2-methylisoborneol hapten.

[0045] Figure 2 This is the standard curve for the 2-MIB indirect competitive ELISA method. Detailed Implementation

[0046] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0047] 2-MIB is an abbreviation for 2-methylisocamphexol; NHS and EDC are abbreviations for N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, respectively; DMF is an abbreviation for N,N-dimethylformamide; THF is an abbreviation for tetrahydrofuran. 2-Methylisoborneol (Catalog No.: ODOR-02S) was purchased from Beijing Bailingwei Technology Co., Ltd.; allyl magnesium bromide (Catalog No.: A110229) was purchased from Haialin Biochemical Technology Co., Ltd.; D-camphor (Catalog No.: D804811), silica gel for column chromatography (Catalog No.: C820912 and C820913), and mercaptoacetic acid (Catalog No.: T818950) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; ethyl acetate (10009418), ethanol (XW00641752), anhydrous sodium sulfate (10020518), dichloromethane (800473660), petroleum ether (80098618), dimethylformamide (81007718), etc. were purchased from Sinopharm Chemical Reagent Co., Ltd. Hemocyanin (H7017), Freund's complete adjuvant (F5881), and Freund's incomplete adjuvant (F5506) were all purchased from Merck Co., Ltd. Goat anti-mouse enzyme-labeled secondary antibody (11-035-008) was purchased from Jackson ImmunoResearch Inc. Balb / c mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Microplates (96-well removable) were purchased from Dimo ​​(Beijing) Biotechnology Co., Ltd.

[0048] Unless otherwise specified, the PBS buffer used in the examples is a pH 7.4, 0.01 M PBS buffer (containing 0.15 mol / L NaCl); PBST is the above PBS buffer solution containing 0.05% Tween-20.

[0049] Example 1: Synthesis and Identification of Hapten

[0050] I. Synthesis of Haptens

[0051] 1. Under nitrogen protection, add 20 mL of allyl magnesium bromide diethyl ether solution (1 mol / L) to a three-necked flask (100 mL) equipped with a thermometer, and freeze at -20±1 °C.

[0052] 2. Using a syringe, slowly add 1520 mg of D-camphor (CAS: 464-49-3) dissolved in 10 mL of tetrahydrofuran into the above solution, and react at -20±5 ℃ for 5 h;

[0053] 3. Quench the Grignard reaction with 10 mL of pure water, then extract with ethyl acetate, collect the organic phase, dry it with anhydrous Na2SO4, filter it, and concentrate it under vacuum to obtain 3 g of oil.

[0054] 4. Dissolve in dichloromethane, add 6g of silica gel (100-200 mesh) and mix. Prepare a chromatography column with silica gel (200-300 mesh) for separation and purification. Elute with petroleum ether: ethyl acetate (5:1) and collect the oily main product 2-allyl isoborneol, about 1.746 g.

[0055] 5. Take the prepared 2-allyl isoborneol (777.2 mg) and add it to a 100 mL flask. Add 20 mL of ethanol and stir until dissolved. Then add 460 μL of mercaptoacetic acid and stir at 60 °C for 4 h.

[0056] 6. Ethanol was removed by vacuum distillation to obtain approximately 1329 mg of oily substance. 2.6 g of silica gel (100-200 mesh) was added and mixed. A silica gel (200-300 mesh) column was prepared for separation and purification. Petroleum ether:ethyl acetate = 5:1 was used as elution, and approximately 916 mg of carboxyl-modified 2-methylisoborneol hapten (Formula 1) was collected.

[0057] II. Identification of Haptens

[0058] The obtained product, 2-methylisoborneol hapten, was identified by mass spectrometry. The identification results are as follows: Figure 1 As shown, a distinct molecular ion peak can be observed at m / z 309, consistent with the molecular weight of the target compound in formula (Ⅰ) (ES). + : M+Na).

[0059] Example 2 Preparation of Artificial Antigen

[0060] The hapten shown in formula (I) was coupled to the carrier protein KLH by the carbodiimide method. The resulting conjugate was used as an immunogen and named 2-MIB-KLH. Its structure is shown in formula (II).

[0061] Formula (II)

[0062] I. Preparation of Immunogens

[0063] 1. Weigh 18 mg of the artificial hapten shown in formula (Ⅰ), dissolve it in 1 mL of DMF to obtain a hapten solution;

[0064] 2. Add 20 mg each of EDC and NHS to the hapten solution prepared in step 1, place it on a rotary mixer (200 rpm), and react at room temperature for about 2.5 h;

[0065] 3. Dissolve 50 mg of KLH protein solution in 3.5 mL of PBS buffer, vortex at 200 rpm for 10 min to ensure complete dissolution, and obtain the carrier protein solution;

[0066] 4. Add the solution from step (2) that has completed the carboxyl activation reaction to the protein solution prepared in step (3), and shake at 200 rpm for 24 h;

[0067] 5. Dialyze the reaction solution obtained in step (4) in a dialysis bag with a molecular weight cutoff of 7-10 kDa at 2-8°C for 72 hours, changing the buffer every 12 hours.

[0068] Further, in step (1), the concentration of the artificial hapten shown in formula (I) is 15-20 mg / mL; in step (2), the carboxyl activator is a combination of EDC and NHS (molar ratio of 1-2:1-2); the mass ratio of the carboxyl activator to the artificial hapten shown in formula (I) is 1.2:1; step (2) is carried out at room temperature and stirred at 200 rpm for 2-3 h; in step (3), the volume-to-mass ratio of PBS buffer to carrier protein is 3 mL:60 mg; in step (4), the mass ratio of formula (I) in the reaction solution of step (2) to KLH in the solution of step (3) is 1:3.

[0069] II. Synthesis of Coated Genes

[0070] 1. Weigh 18 mg of the artificial hapten shown in formula (Ⅰ), dissolve it in 1 mL of DMF to obtain a hapten solution;

[0071] 2. Add 20 mg each of EDC and NHS to the hapten solution prepared in step 1, place it on a rotary mixer (200 rpm), and react at room temperature for about 2.5 h;

[0072] 3. Dissolve 50 mg of OVA protein solution in 3.5 mL of PBS buffer, vortex at 200 rpm for 10 min to ensure complete dissolution, and obtain the carrier protein solution;

[0073] 4. Add the solution from step (2) that has completed the carboxyl activation reaction to the protein solution prepared in step (3), and shake at 200 rpm for 24 h;

[0074] 5. Dialyze the reaction solution obtained in step (4) in a dialysis bag with a molecular weight cutoff of 7-10 kDa at 2-8°C for 72 hours, changing the buffer every 12 hours.

[0075] Further, in step (1), the concentration of the artificial hapten shown in formula (I) is 15-20 mg / mL; in step (2), the carboxyl activator is a combination of EDC and NHS (molar ratio of 1-2:1-2); the mass ratio of the carboxyl activator to the artificial hapten shown in formula (I) is 1.2-1.5:1; step (2) is carried out at room temperature and stirred at 200-300 rpm for 2-3 h; in step (3), the volume-to-mass ratio of PBS buffer to carrier protein is 3-4 mL: 45-75 mg; in step (4), the mass ratio of formula (I) in the reaction solution of step (2) to OVA in the solution of step (3) is 1.5-2.5:6-8.

[0076] Example 3 Preparation of Monoclonal Antibodies

[0077] 1. Animal immunization

[0078] The immunogen prepared in Example 2 was dissolved in physiological saline at a concentration of 100 μg / mouse (1 mg / mL) and mixed with an equal volume of Freund's complete adjuvant. Eight 6-8 week old Balb / c female mice were immunized by subcutaneous injection in the neck and back. On days 7, 14, and 28 after the initial immunization, the immunogen was mixed with an equal volume of Freund's incomplete adjuvant and administered as a booster immunization. Three days before fusion, a booster immunization was administered with 100 μg / mouse of immune complex without Freund's adjuvant.

[0079] 2. Cell fusion and cloning

[0080] Spleen cells from immunized mice were mixed with myeloma cells (SP2 / 0) in the logarithmic growth phase. Then, preheated fusion agent (PEG4000) was slowly added over 30 seconds to induce fusion. The cells were then suspended in HAT medium and a suitable amount of feeder cells were added. The mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 7 days, the medium was partially replaced with HT medium, and after 9 days, the medium was completely replaced.

[0081] After cell fusion, hybridoma cells were screened using a stepwise selection method after 7-10 days of cell growth. Initial selection was performed using an indirect ELISA method (coating agent 2-MIB-OVA, diluted 1:5000 with 0.01M PBS), selecting 534 positive wells. These positive wells were then further screened using an indirect competitive ELISA method. Wells with strong positive results and high inhibition rates were selected for subclonal selection. After multiple subclonal selections, the positive rate reached 100%, ultimately yielding multiple hybridoma cell lines capable of secreting 2-MIB monoclonal antibodies. Among them, cell line 12C10 exhibited the highest antibody titer and sensitivity, with an IC50 value of [missing value]. 50 It was 0.68 ng / mL.

[0082] 3. Preparation and purification of antibodies

[0083] 2-MIB monoclonal antibody was prepared using an in vivo ascites induction method in mice. 8-10 week old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse, followed by intraperitoneal injection of 1-2 × 10⁻⁶ hybridoma cells 7-10 days later. 5 / mouse, ascites fluid was extracted from mice 7-10 days later, centrifuged at 5000 rpm for 10 min at 4℃ to remove the upper oil and lower precipitate, and then purified by the caprylic acid-saturated ammonium sulfate method to obtain 2-MIB monoclonal antibody, which was stored at -20℃.

[0084] The purification method for the caprylic acid-saturated ammonium sulfate monoclonal antibody is as follows:

[0085] ① Take 2 mL of centrifuged ascites fluid and add 4 mL of 0.06 M pH 4.0 acetate buffer solution, then adjust the pH to 4.5 with NaOH; ② The ratio of caprylic acid to diluted ascites fluid is 10 μL: 1 mL. Add 65 μL of caprylic acid dropwise with stirring at room temperature, completing the addition within 30 min. Let stand at 4 ℃ for 2 h, then centrifuge at 10000 rpm for 30 min; ③ Take the supernatant and adjust the pH to 7.4 with NaOH or HAc while stirring. Add saturated ammonium sulfate to 45% saturation at 4 ℃, react for 30 min, and let stand for 2 h; ④ Centrifuge at 10000 rpm for 30 min; ⑤ Dissolve the precipitate in an equal volume of 0.01 M PBS (pH 7.4), dialyze at 4 ℃ for 2 days, changing the dialysate twice a day; ⑥ Centrifuge at 4000 rpm for 10 min, collect the supernatant; after measuring the antibody concentration, store at -20 ℃ for later use.

[0086] 4. Determination of monoclonal antibody concentration

[0087] Using 0.01 M PBS as a blank control, the OD of the purified 2-MIB monoclonal antibody solution was measured using a NanoDrop 2000 UV spectrophotometer. 280 nm OD260 nm Then calculate using the following formula:

[0088] Protein concentration (mg / mL) = 1.45 × OD 280 nm - 0.74 × OD 260 nm

[0089] Results: The concentration of the 2-MIB monoclonal antibody prepared in this invention was 2.05 mg / mL.

[0090] Example 4: Establishment of the 2-MIB Indirect Competitive ELISA Method

[0091] The optimal amounts of coating antigen, 2-MIB monoclonal antibody (12C10), and coating solution were determined using a checkerboard method. The specific procedures are as follows:

[0092] 1) Antigen coating: The 2-MIB-OVA antigen was diluted to a series of concentrations (1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000) with coating buffer, and 100 μL was added to each well of a 96-well plate and incubated at 37°C for 2 h. Two coating buffers were used for screening: 0.01 M phosphate buffer (pH 7.2) and 0.05 M carbonate buffer (pH 9.6).

[0093] 2) Washing: Pour out the liquid in the wells, wash 3 times with washing solution (PBST), 270 μL / well, and pat dry on absorbent paper;

[0094] 3) Blocking: Add 200 μL of blocking solution per well, incubate at 37℃ for 2 h, wash 3 times with washing buffer (PBST) 270 μL per well after blocking, and pat dry on absorbent paper;

[0095] 4) Sample addition: Add 50 μL / well of a series of concentrations of 2-MIB standards (0 ng / mL, 0.1 ng / mL, 0.3 ng / mL, 0.9 ng / mL, 2.7 ng / mL, 8.1 ng / mL and 24.3 ng / mL), then add 50 μL / well of appropriately diluted 2-MIB monoclonal antibody (12C10), and incubate at 37℃ for 30 min; the dilution gradient of the 2MIB monoclonal antibody (12C10) to be tested includes: 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000;

[0096] 5) Washing: Discard the liquid in the wells, wash 3 times with washing buffer (PBST), 270 μL / well, and pat dry on absorbent paper;

[0097] 6) Horseradish peroxidase-labeled secondary antibody: Add 100 μL / well of HRP-goat anti-mouse IgG (1:5000 dilution) and incubate at 37 ℃ for 30 min;

[0098] 7) Washing: Pour out the liquid in the well, wash 3 times with washing solution, 270 μL / well, and pat dry on absorbent paper.

[0099] 8) Color development: Add freshly prepared TMB substrate solution, 100 μL / well, and develop color for 10 min in the dark.

[0100] 9) Termination: Add stop solution, 50 μL / well.

[0101] 10) Measurement: Read the OD450 nm value of each well using an ELISA reader (dual wavelength: 630 nm is the reference filter wavelength).

[0102] Results: The optimal coating solution was determined using the checkerboard method: 0.05 M carbonate buffer (pH 9.6). The optimal dilution ratio for coating the original TCS-OVA was 1:9000, and the optimal dilution ratio for the triclosan monoclonal antibody was 1:27000. Figure 2 This is the standard curve for the 2-MIB indirect competitive ELISA method, with an IC50 of 0.42 ng / mL.

[0103] Example 5: Specificity test of 2-MIB monoclonal antibody

[0104] The cross-reactivity rate of the monoclonal antibody prepared in this invention to 2-MIB structural / functional analogues was tested using the indirect competitive ELISA method described in Example 4, including: borneol, isoborneol, camphor, camphorquinone, and geosmin. The cross-reactivity data are shown in the table below.

[0105] Cross-reactivity rate (100%) = IC 50(2-MIB) / IC 50（类似物） ×100%

[0106] Table 1. Cross-reactivity data of monoclonal antibody 12C10

[0107]

[0108] It can be seen that the monoclonal antibody obtained from the 2-methylisoborneol hapten compound of the present invention has excellent sensitivity and accuracy for the detection of 2-methylisoborneol, and is suitable for the detection of trace amounts of 2-methylisoborneol in water.

Claims

1. A 2-methylisoborneol hapten, characterized in that, The structural formula is shown in equation (Ⅰ); Equation (Ⅰ).

2. The method for preparing the 2-methylisoborneol hapten according to claim 1, characterized in that, The synthesis route is as follows: 。 3. The preparation method according to claim 2, characterized in that, The method for preparing the 2-methylisoborneol hapten of formula (I) includes the following steps: (S1) Under an inert atmosphere, D-camphor solution (CAS: 464-49-3) was added to allyl magnesium bromide solution and reacted at -30℃ to -10℃. After the reaction was complete, the Grignard reaction in step (S1) was quenched, and the product was extracted, dried, filtered, concentrated, and purified to obtain intermediate (IA). (S2) Under an inert atmosphere, intermediate (IA) reacts with mercaptoacetic acid to give product compound (I).

4. The preparation method according to claim 3, characterized in that, In step (S1), the solvent for the D-camphor solution is selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, ethanol, ethyl acetate, and butyl acetate; the concentration of the D-camphor solution is 0.5-1.5 mol / L, preferably 0.8-1 mol / L; the solvent for the allyl magnesium bromide solution is selected from at least one of diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, and dichloromethane; the concentration of the allyl magnesium bromide solution is 0.5-1.5 mol / L, preferably 0.8-1 mol / L. Preferably, the molar ratio of D-camphor to allyl magnesium bromide is 1:1-3, more preferably 1:1.5-2.

5. The preparation method according to claim 3, characterized in that, In step (S2), the molar ratio of intermediate (IA) to mercaptoacetic acid is 1-1.5:1-1.5, preferably 1-1.1:1-1.

1. The reaction between the alkenyl and mercapto groups is rapid and complete, and the reactants are fed at a molar ratio close to 1:

1. After the reaction in step (S2) is completed, an oily substance is obtained. Separation and purification are carried out by silica gel chromatography, with petroleum ether and ethyl acetate as the eluent at a volume ratio of 3-5:

1.

6. A 2-methylisoborneol artificial antigen, obtained by conjugating the 2-methylisoborneol hapten of claim 1 with a carrier protein; further, the carrier protein is selected from at least one of keyhole hemocyanin, bovine serum albumin, hemocyanin, thyroprotein, and ovalbumin.

7. The method for preparing the 2-methylisoborneol artificial antigen according to claim 6, characterized in that, The steps include: coupling the carrier protein to the carboxyl group of the 2-methylisoborneol hapten shown in formula (Ⅰ) by the carbodiimide method; Specifically, the preparation method of the 2-methylisoborneol artificial antigen includes the following steps: (1) The artificial hapten shown in formula (Ⅰ) is dissolved in DMF to obtain a hapten solution; (2) Take the hapten solution, add the carboxyl activator, and react; (3) The carrier protein solution was dissolved in PBS buffer to obtain the carrier protein solution; (4) Add the solution from step (2) that has completed the carboxyl activation reaction to the protein solution prepared in step (3); (5) Dialyze the liquid phase obtained in step (4) in a dialysis bag with a molecular weight cutoff of 7-10 KDa at 2-8°C for 72-96 h, and change the buffer every 12-14 h.

8. The method for preparing the 2-methylisoborneol artificial antigen according to claim 7, characterized in that, In step (1), the concentration of the artificial hapten shown in formula (I) is 15-20 mg / mL; in step (2), the carboxyl activator is a combination of EDC and NHS (molar ratio of 1-2:1-2); the molar ratio of the carboxyl activator to the artificial hapten shown in formula (I) is 1.2-1.5:1; step (2) is carried out at room temperature and stirred at 200-300 rpm for 2-3 h; in step (3), the volume-to-mass ratio of PBS buffer to carrier protein is 3-4 mL: 45-75 mg; in step (4), the mass ratio of formula (I) in the reaction solution of step (2) to KLH in the solution of step (3) is 1.5-2.5:6-8.

9. A 2-methylisoborneol antibody, characterized in that, The 2-methylisoborneol artificial antigen described in claim 6 is obtained by animal immunization; further, the antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody.

10. Any of the following uses of the 2-methylisoborneol hapten of formula (I) as described in claim 1, or the 2-methylisoborneol artificial antigen as described in claim 6, or the 2-methylisoborneol antibody as described in claim 9: (1) Immunoassay methods / products for the detection of 2-methylisoborneol; (2) Used for the detection of 2-methylisoborneol; Furthermore, the analytical method / product type includes immunochromatography, membrane strips, kits, chips, models, or detection platforms; even further, the analytical method is an indirect competitive enzyme-linked immunosorbent assay (ELISA).

Images