Safrole hapten and application of safrole hapten in immunoassay detection of safrole

Abstract

The invention provides a safrole hapten and application of the safrole hapten in detection of safrole through immunoassay. The structure of the safrole hapten is shown in the specification. The two safrole haptens provided by the invention have spacer arms with proper lengths, and after the safrole haptens are coupled with carrier protein, the haptens can be fully exposed, and recognition of the spacer arms can be avoided as much as possible. The safrole hapten is used for preparing an artificial antigen and an antibody, and the obtained safrole monoclonal antibody is high in titer, strong in specificity and high in affinity. An immunoassay method constructed on the basis of the safrole artificial antigen and antibody has high specificity and sensitivity, the lowest detection limit LOD is 0.09 ng / mL, the half inhibitory concentration IC50 is 2.05 ng / mL, the quantitative detection range is 0.28-14.89 ng / mL, no cross reaction exists on safrole analogues, and rapid qualitative and quantitative detection can be conducted on safrole in a sample.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and particularly relates to a safrole hapten and a preparation method thereof, an artificial antigen, an antibody and application thereof in immunoassay detection of safrole. Background Art

[0002] Safrole (4-allyl-1,2-methylenedioxybenzene, C 10 H 10 Safrole (O2) is a naturally occurring organic compound belonging to the phenylpropene family and a component of volatile oils in many plants. Safrole, often found in liquid form, has a sweet and pungent odor and is widely distributed in plants of the Lauraceae family (e.g., sassafras and camphor trees) and the Piperaceae family.

[0003] Safrole is toxic, can damage the liver, and may be carcinogenic. Animal studies have shown that safrole can cause gene mutations and liver tumors. Safrole levels can increase dramatically in spoiled spice plants.

[0004] Currently, the commonly used methods for detecting safrole include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). These methods suffer from complex pretreatment, expensive instrumentation, and high costs, and they cannot achieve efficient and rapid detection and monitoring of safrole. Furthermore, the detection limit of currently reported safrole immunoassays is only 200 ng / mL, indicating low sensitivity. Therefore, it is necessary to develop a rapid method for detecting safrole that is simple to operate, highly sensitive, and accurate. Summary of the Invention

[0005] In order to solve all or part of the above technical problems, the present invention provides the following technical solutions: The first aspect of the present invention provides a safrole hapten, the structure of which is shown in formula (I) or formula (II): .

[0006] The two safrole haptens provided by the present invention have spacer arms of appropriate lengths, which, after being coupled to a carrier protein, can fully expose the hapten while avoiding recognition of the spacer arm as much as possible, thereby generating better antibodies to recognize safrole. If the spacer arm of the hapten is short, it is easy for the carrier protein to mask the characteristic structure of the analyte, resulting in a poor antibody response; if the spacer arm is long, it is easy for the conformation of the hapten molecule to fold, thereby being masked by the carrier protein, and easily generating antibodies against the spacer arm. The safrole hapten of the above structure provided by the present invention has a spacer arm of a certain length connected between the hapten target epitope and the carrier protein, thereby keeping the characteristic structure of the target molecule away from the carrier protein, maximizing the exposure of the hapten to the immune system.

[0007] The second aspect of the present invention provides a method for preparing the safrole hapten described in any one of the above technical solutions.

[0008] The preparation method of the safrole hapten represented by formula (II) comprises: A first mixed reaction system containing eugenol, pyridine and aluminum halide is subjected to a first reaction at 100-120° C. to obtain 4-allylcatechol; allowing a second mixed reaction system containing the 4-allylcatechol, methyl dichloroacetate, and an acid-binding agent to undergo a second reaction at 80-100° C. to obtain methyl 5-allylbenzo[D][1,3]dioxolane-2-carboxylate; The 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid methyl ester is hydrolyzed under alkaline conditions, and then the pH value of the hydrolysis reaction product is adjusted to acidic to obtain 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid, i.e., the safrole hapten shown in formula (II).

[0009] In some embodiments, the molar equivalents of eugenol, pyridine, and aluminum halide in the first mixed reaction system satisfy 1:1-3:1-2. The aluminum halide includes but is not limited to aluminum trichloride.

[0010] In some embodiments, the first reaction time is 10-12 hours.

[0011] In some embodiments, the first mixed reaction system further includes a solvent, and the solvent includes one or a combination of more of acetonitrile, dimethylformamide, and dimethyl sulfoxide, but is not limited thereto.

[0012] In some embodiments, the molar equivalents of 4-allylcatechol, methyl dichloroacetate, and the acid-binding agent in the second mixed reaction system satisfy 1:1-3:1-3. The acid-binding agent includes but is not limited to potassium carbonate.

[0013] In some embodiments, the second mixed reaction system further includes a solvent, which can be any aprotic solvent known in the art, including but not limited to acetonitrile.

[0014] In some embodiments, the second reaction time is 6 to 8 hours.

[0015] In some embodiments, the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid methyl ester and a base are mixed in a molar equivalent ratio of 1:1-3, and the hydrolysis is performed at 0-40°C.

[0016] In some implementations, the hydrolysis is performed by adding the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid methyl ester and a base to an aqueous solution of 1,4-dioxane.

[0017] In some embodiments, the pH of the hydrolyzed reaction product is adjusted to 2-4.

[0018] The method for preparing the safinin hapten as shown in formula (I) comprises: The 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid is mixed with N,N'-carbonyldiimidazole, and then tert-butyl aminobutyrate is added, and a fourth reaction is carried out at 0-40℃ to obtain tert-butyl (5-allylbenzo[D][1,3]dioxolane-2-carbonyl) aminobutyrate; The tert-butyl (5-allylbenzo[D][1,3]dioxolane-2-carbonyl) aminobutyrate is mixed with an acid and a fifth reaction is carried out at 0-40℃ to obtain (5-allylbenzo[D][1,3]dioxolane-2-carbonyl) aminobutyric acid, i.e. the safinin hapten as shown in formula (I).

[0019] The preparation method of the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid is the same as above.

[0020] In some embodiments, the molar equivalent of the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid, N,N'-carbonyldiimidazole, and tert-butyl aminobutyrate satisfies 1:1-2:1-2.

[0021] In some embodiments, the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid is first mixed with N,N'-carbonyldiimidazole for 0.5-2h, and then the tert-butyl aminobutyrate is added for 2-4h.

[0022] In some embodiments, the fourth reaction is carried out in a solvent, and the solvent comprises dichloromethane, but is not limited thereto.

[0023] In some embodiments, the molar equivalent of the tert-butyl (5-allylbenzo[D][1,3]dioxolane-2-carbonyl) aminobutyrate and the acid satisfies 1:1-3.

[0024] In some embodiments, the fifth reaction is carried out in a solvent, and the solvent comprises 1,4-dioxane, but is not limited thereto.

[0025] In some embodiments, the time of the fifth reaction is 4-6h.

[0026] The third aspect of the present application provides a safinin artificial antigen, which is obtained by coupling the safinin hapten with a carrier protein, and the structure of the safinin artificial antigen is shown in formula (III) or formula (IV): ; In the formula, Protein is a carrier protein.

[0027] The artificial antigen of safinin with the structure as shown in formula (III) is obtained by coupling the carrier protein with the hapten of safinin with the structure as shown in formula (I). For example, the carrier protein is coupled by active ester method.

[0028] The artificial antigen of safinin with the structure as shown in formula (IV) is obtained by coupling the carrier protein with the hapten of safinin with the structure as shown in formula (II). For example, the carrier protein is coupled by active ester method.

[0029] In some embodiments, the carrier protein is any one of bovine serum albumin, chicken egg albumin, hemocyanin, lactoferrin, but not limited thereto.

[0030] In some embodiments, the artificial antigen of safinin has the structure as shown in formula (III) and the carrier protein is bovine serum albumin.

[0031] In some embodiments, the artificial antigen of safinin has the structure as shown in formula (IV) and the carrier protein is chicken egg albumin.

[0032] The fourth aspect of the present application provides a combination of artificial antigens of safinin, comprising an immunogen and a coating antigen, wherein the immunogen is the artificial antigen of safinin with the structure as shown in formula (III) and the carrier protein is bovine serum albumin, and the coating antigen is the artificial antigen of safinin with the structure as shown in formula (IV) and the carrier protein is chicken egg albumin.

[0033] The fifth aspect of the present application provides a safinin antibody, which is obtained by immunizing an animal with the artificial antigen of safinin according to any one of the technical solutions.

[0034] In some embodiments, the safinin antibody is a monoclonal antibody.

[0035] In some embodiments, the safinin antibody is obtained by immunizing an animal with the artificial antigen of safinin with the structure as shown in formula (III) and the carrier protein is bovine serum albumin.

[0036] The sixth aspect of the present application provides the use of the hapten of safinin according to any one of the technical solutions, the artificial antigen of safinin according to any one of the technical solutions, the combination of artificial antigens of safinin, or the safinin antibody according to any one of the technical solutions in the preparation of a safinin detection product or in the detection of safinin for the purpose of non-disease treatment diagnosis.

[0037] The seventh aspect of the present application provides a safinin detection device, comprising the artificial antigen of safinin according to any one of the technical solutions and the safinin antibody according to any one of the technical solutions.

[0038] In some embodiments, the safinin detection device comprises a test strip assembly or an ELISA kit.

[0039] In some embodiments, the test strip assembly includes a test strip and a reaction cup, the test strip contains a test line and a control line, the test line is coated with the safrole artificial antigen, the control line is coated with a goat anti-mouse monoclonal antibody, and the reaction cup contains the safrole antibody labeled with colloidal gold.

[0040] In some embodiments, the test strip includes a base plate, and a sample pad, a cellulose membrane, and a water-absorbing pad sequentially arranged on the base plate, and the detection line and the control line are arranged on the cellulose membrane.

[0041] During the test, the sample to be tested is added to the reaction cup, and is fully mixed and incubated with the colloidal gold-labeled safrole antibody therein. The mixture is then dripped onto the absorbent pad of the test strip and allowed to stand until the mixture fully penetrates the absorbent pad, and the result is determined.

[0042] The judgment method is: if the control line does not show color, the test effect is invalid and needs to be retested; if the control line shows color, the test effect is valid, and the test line is further interpreted: if the test line does not show color or the color is very weak, it means that the sample to be tested contains safrole, and the judgment result is positive or weakly positive; if the test line shows color, it means that the sample to be tested does not contain safrole, and the judgment result is negative.

[0043] In some embodiments, in the safrole detection device, the structure of the safrole artificial antigen is as shown in formula (IV) and the carrier protein is chicken ovalbumin, and the safrole antibody is obtained by immunizing an animal with a safrole artificial antigen having a structure as shown in formula (III) and a carrier protein of bovine serum albumin.

[0044] In some embodiments, in the safrole detection device, the safrole antibody is a monoclonal antibody.

[0045] An eighth aspect of the present invention provides an immunoassay method for detecting safrole, which uses the safrole artificial antigen described in any technical solution of the present invention and the safrole antibody described in any technical solution for detection.

[0046] In some embodiments, the structure of the safrole artificial antigen is as shown in formula (IV) and the carrier protein is chicken ovalbumin, and the safrole antibody is obtained by immunizing an animal with a safrole artificial antigen having a structure as shown in formula (III) and a carrier protein of bovine serum albumin.

[0047] In some embodiments, the safrole antibody is a monoclonal antibody.

[0048] In some embodiments, the immunoassay method is colloidal gold immunochromatography or ELISA.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention provides two safrole haptens with spacer arms of appropriate lengths. After being coupled to a carrier protein, the haptens can be fully exposed while minimizing the recognition of the spacer arms, thereby generating better antibodies that recognize safrole.

[0050] (2) The present invention uses the safrole hapten to prepare safrole artificial antigens and antibodies, and the obtained safrole monoclonal antibodies have high titer, strong specificity and high affinity.

[0051] (3) The immunoassay constructed based on the artificial antigen and antibody for safrole provided by the present invention has good specificity for detecting safrole, has no cross-reaction with safrole analogs, and has high sensitivity. It can perform rapid qualitative and quantitative detection of safrole in samples, is easy to operate, and produces accurate and reliable test results.

[0052] (4) The minimum detection limit (LOD) of the ELISA method constructed based on the artificial antigen and antibody of safrole provided by the present invention is 0.09 ng / mL, and the half inhibitory concentration (IC) is 50 The concentration of β-catenin was 2.05 ng / mL, and the quantitative detection range was 0.28~14.89 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 This is a UV full wavelength scanning identification diagram of BSA, SF-3, and SF-3-BSA in the embodiment of the present invention; Figure 2 This is a full-wavelength ultraviolet scanning identification diagram of OVA, SF-4, and SF-4-OVA in the embodiment of the present invention; Figure 3 This is a standard curve diagram of the ELISA method constructed using safrole monoclonal antibodies prepared using SF-4-OVA as the coating agent and SF-3-BSA as the immunogen in the examples of the present invention; Figure 4 Schematic diagram of the structure of the colloidal gold test strip provided in Example 5 of the present invention; Figure 5 This is a diagram showing the test results of safrole detection using a colloidal gold test strip in Example 5 of the present invention. DETAILED DESCRIPTION

[0055] The technical solutions of the present invention are described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand and implement the technical solutions of the present invention. The specific functional details disclosed herein should not be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching those skilled in the art to employ the present invention in various ways in virtually any appropriate detailed embodiment.

[0056] In addition, unless otherwise specified, the various raw materials used in the following examples can be purchased from the market and other channels, the various production and testing equipment used are also equipment known in the art, and the testing methods used are also methods known in the art.

[0057] Unless otherwise specified, the amounts of raw materials in the examples of the present invention expressed in equivalents (eq) refer to molar equivalents.

[0058] Example 1 This example provides a safrole hapten and a method for synthesizing the same, and identifies the synthesized safrole hapten. 1. Synthesis of safrole hapten SF-3 The synthetic route of safrole hapten SF-3 is as follows:

[0059] The specific steps are as follows: (1) 1.2 eq of aluminum chloride and 1.2 eq of pyridine were dissolved in acetonitrile. After the exotherm disappeared, 1 eq of eugenol was added and the reaction was refluxed overnight. After the reaction was completed, the reaction solution was poured into an ice-cold hydrochloric acid solution, extracted with ethyl acetate and water, and the organic phase was retained. 15 g of silica gel powder was added to the organic phase and dried using a rotary evaporator. 4-allyl catechol was purified by column chromatography using a developing solvent (volume ratio, petroleum ether: ethyl acetate: formic acid: 70:30:1) to obtain 4-allyl catechol.

[0060] (2) 1 eq of 4-allyl catechol, 1.2 eq of methyl dichloroacetate and 1.2 eq of potassium carbonate were added into acetonitrile and refluxed overnight; after the reaction was completed, the organic phase was reserved after extraction with ethyl acetate and water; 15 g of silica gel powder was added into the organic phase and then rotary evaporation was used to dry it; 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid methyl ester was obtained by column chromatography using developing agent (volume ratio, petroleum ether: ethyl acetate: formic acid: 80:20:1); 1 eq of 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid methyl ester and 2 eq of sodium hydroxide were added into 1,4-dioxane aqueous solution and reacted for 3 h; after the reaction was completed, hydrochloric acid was added to adjust the pH value to 4; the organic phase was reserved after extraction with ethyl acetate and water, and then rotary evaporation was used to dry the organic phase to obtain 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid.

[0061] (3) 1 eq of 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid and 1 eq of N,N'- carbonyldiimidazole were added into dichloromethane and reacted for 1 h, and then tert-butyl aminobutyrate was added and reacted for 3 h; after the reaction was completed, the organic phase was reserved after extraction with ethyl acetate and water; 15 g of silica gel powder was added into the organic phase and then rotary evaporation was used to dry it; (5-allyl benzo[D][l,3]dioxol-2-carbonyl) tert-butyl aminobutyrate was obtained by column chromatography using developing agent (volume ratio, petroleum ether: ethyl acetate: 80:25); 1 eq of (5-allyl benzo[D][l,3]dioxol-2-carbonyl) tert-butyl aminobutyrate and 3 eq of hydrochloric acid were added into 1,4-dioxane and reacted for 4 h; after the reaction was completed, (5-allyl benzo[D][l,3]dioxol-2-carbonyl) aminobutyric acid was obtained by rotary evaporation, which was safrole hapten SF-3.

[0062] 2. Identification of safrole hapten SF-3 The result of nuclear magnetic resonance hydrogen spectrum of safrole hapten SF-3 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 11.77(s, 1H), 8.33 (t, J = 4.9 Hz, 1H), 6.97 – 6.65 (m, 3H), 6.34 (s, 1H), 5.94(tt, J = 9.4, 7.5 Hz, 1H), 5.19 – 4.83 (m, 2H), 3.57 – 2.94 (m, 4H), 2.33 (t, J = 8.8 Hz, 2H), 1.80 (tt,J = 9.0, 5.8 Hz, 2H). The mass spectrometry results of safrole hapten SF-3 are: MS: 291, ESI-[MH] + :290.

[0063] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results correspond to the molecular weight of safrole hapten, and the hydrogen spectrum number of nuclear magnetic resonance corresponds to the hydrogen spectrum number on the backbone structure of safrole hapten, indicating that safrole hapten was successfully prepared and recorded as SF-3. Its structural formula is shown in formula (I):

[0064] 3. Synthesis of safrole hapten SF-4 The synthetic route of safrole hapten SF-4 is as follows:

[0065] Safrole hapten SF-4 is the precursor structure 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid obtained in the process of preparing safrole hapten SF-3. The preparation method thereof has been described in detail above and will not be repeated here.

[0066] 4. Identification of safrole hapten SF-4 The results of the nuclear magnetic resonance hydrogen spectrum of safrole hapten SF-4 are: 1 H NMR (600 MHz, DMSO- d 6) δ 11.48(s, 1H), 7.04 – 6.65 (m, 3H), 6.39 (s, 1H), 5.94 (tt, J = 9.4, 7.5 Hz, 1H), 5.17 – 4.81 (m, 2H), 3.88 – 2.98 (m, 2H). The mass spectrometry results of safrole hapten SF-4 are: MS: 206, ESI-[MH] + :205.

[0067] It can be seen from the mass spectrometry and nuclear magnetic resonance results that the mass spectrometry results correspond to the molecular weight of safrole hapten, and the hydrogen spectrum number of nuclear magnetic resonance corresponds to the hydrogen spectrum number on the backbone structure of safrole hapten, indicating that safrole hapten was successfully prepared and recorded as SF-4. Its structural formula is shown in formula (II):

[0068] Example 2 This example provides a safrole artificial antigen and a synthesis method thereof, and identifies the synthesized safrole artificial antigen.

[0069] 1. Synthesis of safrole artificial antigen (1) The safrole hapten SF-3 (3.156 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, and EDC (3.5 mg, 0.018 mol) and NHS (2.1 mg, 0.018 mmol) were added, dissolved in 100 μL DMF, and stirred at room temperature for 3 h to obtain a hapten activation solution; (2) Dissolve BSA (bovine serum albumin, 10 mg, 0.00015 mol) in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain a BSA solution. Stir the BSA solution and hapten activation solution at 4 °C overnight. (3) Collect the reaction solution, transfer it to a treated dialysis bag, and then place it in 5 L of 0.01 M PBS buffer; dialyze at 4 °C for 3 days, and replace the solution with pre-cooled 0.01 M PBS buffer every 8 hours; (4) After purification by dialysis, the artificial antigen of safrole was obtained, which was named SF-3-BSA. After concentration determination and UV spectroscopy identification, it was stored in a -20 ℃ refrigerator.

[0070] The safrole artificial antigen was prepared according to the same method as above, except that OVA (chicken ovalbumin) was used instead of BSA and SF-4 was used instead of SF-3, and was recorded as SF-4-OVA.

[0071] 2. Identification of safrole artificial antigen BSA, OVA, SF-3, SF-4, SF-3-BSA and SF-4-OVA were scanned and identified using the ultraviolet full wavelength method (200-350nm).

[0072] Figure 1 This is the UV full wavelength scanning identification diagram of BSA, SF-3, and SF-3-BSA, such as Figure 1 As shown in the figure, by comparing the maximum absorbance values ​​of each substance before and after conjugation, the absorption curve of SF-3-BSA is significantly different from that of the carrier protein BSA. SF-3 exhibits a strong absorption peak above 343 nm. However, after conjugation with BSA, the absorption peak of SF-3-BSA is close to that of BSA at 230 nm and significantly higher than that of BSA at 280 nm. Furthermore, the curve relative to the hapten SF-3 is significantly shifted. Since the unreacted components are completely removed by dialysis after conjugation, the characteristic peaks of the conjugation product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex between the carrier protein BSA and SF-3.

[0073] Figure 2are UV full wavelength scanning identification figures of OVA, SF-4, SF-4-OVA, as shown in Figure 2 The absorption curve of SF-4-OVA appears characteristic peaks different from OVA and SF-4, indicating that the reaction product is a complex of carrier protein OVA and SF-4.

[0074] The above results show that the artificial antigens SF-3-BSA and SF-4-OVA of safrole are successfully prepared, and the structural formulae are shown in formula (III) and (IV): ; Among them, Protein is carrier protein BSA or OVA.

[0075] Example 3 The present embodiment provides a monoclonal antibody of safrole and a preparation method thereof.

[0076] 1. Animal immunization SF-3-BSA prepared in Example 2 is used as an immunogen, and SF-3-BSA is diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and emulsified thoroughly, and 8-week-old BALB / c female mice are immunized. When immunized for the first time, 3 BALB / c female mice are vaccinated subcutaneously in multiple sites in the abdomen, and the vaccination dose is 100 μg per mouse, and 0.1 ml per BALB / c female mouse; 14 days later, the second immunization is performed, and the immunogen is emulsified with an equal volume of Freund's incomplete adjuvant, and the immunization dose is the same as that of the first immunization, and the number of booster immunizations is 3. After the immunization is completed, the mouse tail serum is collected. The titer and inhibition rate are determined. The mouse with the best effect is selected for impact immunization, and the vaccination dose is 100 μg per mouse.

[0077] 2. Cell fusion Three days after the sprint immunization, cell fusion is performed according to the conventional PEG (polyethylene glycol, molecular weight 1450) method, and the specific steps are as follows: a. After the mouse is sacrificed by eye blood collection, it is immediately disinfected in 75% alcohol for 5 min or so, and the mouse's spleen is taken out by sterile operation, ground with the rubber head of a syringe, and passed through a 200-mesh cell screen to obtain a spleen cell suspension, which is collected, centrifuged (1000 rpm, 7 min), and washed with RPMI-1640 culture medium three times. After the last centrifugation, the spleen cells are diluted to a certain volume, counted, and reserved; b. Collect SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are cultured in 10% FBS (fetal bovine serum) RPMI1640 culture medium in a 5% CO2 incubator, and the number of SP2 / 0 tumor cells is required to reach (1-4) × 10 7, ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. When fusion occurs, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0078] c. Mix the two cells and adjust the volume to 15 mL. Centrifuge at 1000 rpm / min for 7 minutes and discard the supernatant. Remove the centrifuge tube and shake it in a 60° medium-speed circular motion. Then prepare for fusion. The fusion time is controlled as follows: First minute: slowly add PEG; Second minute: Continue to rotate for 1 minute; Third minute: add 1 mL of basal culture medium; Fourth minute: add 3 mL of basal culture medium; Fifth minute: add 8 mL of basal culture medium; Sixth minute: add 8 mL of basal culture medium; After fusion, the cell suspension was centrifuged (1000 rpm / min, 7 min), and the incubated 200 mL HAT medium was taken out and placed in a clean bench for use.

[0079] d. After centrifugation, discard the supernatant of the hybridoma cells, pipette 2 mL of HAT medium into a centrifuge tube, and gently pipette to evenly distribute the cells. Then transfer the cell solution in the centrifuge tube to 200 mL of HAT medium. Gently shake the medium to evenly disperse the cells, and then plate the cells.

[0080] 3. Cell screening and cell line establishment On the third day of cell fusion, the fused cells were subjected to a half-change of RPMI-1640 screening medium. On the fifth day, the medium was fully changed with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 2% 50×HT. On the seventh day, the cell supernatant was collected for screening. The screening process was divided into two steps: first, positive cell wells were screened using the ic-ELISA method; second, safrole was used as a standard and the inhibitory effect of the positive cells was determined using the ic-ELISA method; cell wells with good inhibition of the safrole standard were selected and subcloned using the limiting dilution method, and then tested using the same method seven days later; three subcloning steps were performed according to the above method to finally obtain the safrole monoclonal antibody cell line SF-Q2C.

[0081] 4. Preparation and identification of safrole monoclonal antibodies 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, 1×10 6Starting on the seventh day of incubation, ascites fluid was collected from safrole hybridoma cells and purified by immunoaffinity chromatography. Under neutral conditions, protein G binds to IgG immunoglobulins in the ascites, allowing other impurities to flow through and be discarded. IgG and protein G were then desorbed under acidic conditions using 0.01 M glycine buffer, yielding highly pure IgG immunoglobulins. The IgG immunoglobulin solution was adjusted to a neutral pH using 0.01 M Tris-HCl buffer and then dialyzed for desalination, ultimately yielding highly pure safrole monoclonal antibodies. The purified monoclonal antibodies were stored at -20°C.

[0082] 5. Sensitivity evaluation of safrole monoclonal antibodies (indirect competitive ELISA) SF-4-OVA prepared in Example 2 was used as the coating agent and diluted with coating solution (0.05 M carbonate buffer solution, pH 9.6) to a concentration of 500 ng / mL. 100 μL / well was used to coat a 96-well ELISA plate and incubated at 4°C overnight (12 h). Discard the coating solution, wash twice with PBST (0.01 M PBS, 0.06% v / v Tween-20), and pat dry; Add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well and block at 37°C for 1 h; Discard the blocking solution, clap the plate, dry it at 37℃, take it out, and put it in a ziplock bag for later use; The safrole monoclonal antibody prepared using SF-3-BSA as the immunogen was diluted with PBST at a volume ratio of 1:128,000. The safrole standard was diluted with PBST to different concentrations, obtaining safrole standard dilutions with concentrations of 10,000 ng / mL, 1,000 ng / mL, 125 ng / mL, 70 ng / mL, 50 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, and 0.001 ng / mL. Add 50 μL / well of safrole standard dilutions of different concentrations (three sets in parallel), then add 50 μL / well of diluted safrole monoclonal antibody, incubate at 37°C for 40 min, wash five times, and pat dry; Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000-fold with PBST), incubate at 37°C for 30 min, wash five times, and pat dry; Add 100 μL / well color development solution and develop for 10 min; The reaction was terminated by adding 50 μL of 10% H2SO4 solution and the OD value was read at 450 nm; ELISA standard curve drawing: B / B0 as the ordinate (B is the absorbance OD of different concentrations of standard sample of safrole 450 , B0 is the absorbance OD of blank control well 450 ), the logarithm of the concentration of safrole standard as the abscissa, the curve fitting is carried out by using Logistic function, and the standard curve is prepared, and the formula of the standard curve is obtained.

[0083] Figure 3 is the standard curve of ELISA method constructed by using SF-4-OVA as coating agent and the safrole monoclonal antibody prepared in the above, as shown in Figure 3 , the minimum detection limit LOD is 0.09 ng / mL, the half inhibition concentration IC 50 is 2.05 ng / mL, and the quantitative detection range is 0.28-14.89 ng / mL.

[0084] 6. Specificity evaluation of safrole monoclonal antibody Dihydro-safrole, piperine and sesamol are safrole analogues, and the specificity of the safrole monoclonal antibody prepared in this embodiment is evaluated by cross reaction experiment.

[0085] According to the above sensitivity evaluation method, the difference is that the safrole standard is replaced by the standards of dihydro-safrole, piperine and sesamol, and the detection is carried out at the same dilution ratio, and the IC50 values of each structural analogue are obtained.

[0086] The cross reaction rate (CR) of safrole is calculated according to the following formula: CR (%) = IC50 (safrole) / IC50 (structural analogue) x 100%, and the smaller the cross reaction rate, the stronger the specificity.

[0087] Table 1. Cross reaction results of safrole monoclonal antibody with safrole and its analogues ; Note: NR means no reaction, and no reaction means that the antibody does not recognize the analogue.

[0088] As shown in Table 1, the cross reaction rate of the safrole monoclonal antibody to safrole is 100 %, the IC 50 is 2.05 ng / mL, and there is no cross reaction to dihydro-safrole, piperine and sesamol. It shows that the antibody for detecting safrole has high recognition ability to safrole, strong specificity, can effectively exclude the interference of methylenedioxybenzene analogues (such as dihydro-safrole, piperine and sesamol), and can specifically detect safrole.

[0089] In conclusion, the prepared monoclone antibody of safrole has excellent detection performance, high sensitivity and strong specificity.

[0090] Example 4 The present embodiment provides a combination of an immunogen and a coating agent of safrole.

[0091] The coating agent of safrole artificial antigen SF-4-OVA prepared in Example 2 is used to evaluate the combination effect of the immunogen and the coating agent by the serum titer and inhibition rate obtained by the indirect competitive ELISA method using the monoclone antibody of safrole prepared in Example 3 with SF-3-BSA as the immunogen. The specific operation steps are as follows: 1. The coating agent is diluted with the coating solution (0.05 M carbonate buffer solution, pH 9.6) to a concentration of 1000 ng / mL, and coated on a 96-well enzyme-labeled plate at an addition amount of 100 μL / well, and then placed in a 4 ℃ refrigerator overnight, and then the coating solution is discarded, and the plate is washed twice with PBST (0.01M PBS, 0.06% Tween-20 (v / v)); 2. 120 μL of blocking solution (PBST containing 7% skimmed milk powder) is added to each well, and then blocked at 37 ℃ for 1 h, and then the blocking solution is discarded, and the plate is dried in a drying oven at 37 ℃ for standby; 3. The monoclone antibody of safrole is diluted with PBST at a volume ratio of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 and 1:256000, and a blank control hole (PBST instead) is set at the same time; 1 mg / mL of the standard sample of safrole is diluted 1000 times with PBST to obtain a standard sample diluent of safrole with a concentration of 1 μg / mL; 4. The titer column is set as follows: 50 μL of PBST is added to each well, and then 50 μL of the monoclone antibody of safrole with different dilution ratios is added to each well, and then 50 μL of PBST is added to the last hole instead of the antibody; 5. The inhibition column is set as follows: 50 μL of the standard sample diluent of safrole is added to each well, and then 50 μL of the monoclone antibody of safrole with different dilution ratios is added to each well, and then 50 μL of PBST is added to the last hole instead of the antibody; 6. Incubate at 37 ℃ for 40 min, and then wash the plate 5 times; 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), and then incubate at 37 ℃ for 30 min, and then wash the plate 5 times; 8. Add color developing solution, and then incubate at 37 ℃ for 10 min; 9. Add 10% v / v H2SO4 to terminate the reaction, and then read the OD value at 450 nm;450 The inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition x 100%, when the antibody dilution factor corresponding to the inhibition rate of about 1.0.

[0092] The combination results of the safrole immunogen and the coating agent are shown in Table 2.

[0093] The present application also uses the same method as described above to couple SF-4 with lactoferrin (LF), SF-3 with OVA, and SF-3 with LF to prepare artificial antigens SF-4-LF, SF-3-OVA, and SF-3-LF, respectively. The combination effects of different immunogens and coating agents are evaluated using the same method as described above, and the results are shown in Table 2.

[0094] Table 2 Combination effects of different immunogens and coating agents ; As shown in Table 2, the monoclonal antibody prepared using the safrole artificial antigen SF-3-BSA as the immunogen is the best combination of immunogens and coating agents with SF-4-OVA, and under this combination, the monoclonal antibody can not only specifically recognize the target analyte safrole, but also has excellent antibody sensitivity.

[0095] Example 5 The present embodiment provides a colloidal gold test strip assembly for detecting safrole.

[0096] 1. Assembly of colloidal gold rapid test strip Figure 4 The structure of the colloidal gold test strip of the present embodiment is shown in Figure 4 As shown in Table 2, the monoclonal antibody prepared using the safrole artificial antigen SF-3-BSA as the immunogen is the best combination of immunogens and coating agents with SF-4-OVA, and under this combination, the monoclonal antibody can not only specifically recognize the target analyte safrole, but also has excellent antibody sensitivity.

[0097] The coating agent (SF-4-OVA) was sprayed on the NC membrane with a spraying amount of 0.8 μL / cm using an XYZ three-dimensional spray point membrane drawing instrument, as the test line (T line); the goat anti-mouse IgG was sprayed on the NC membrane with the same method and dosage, as the control line (C line); the test line (T line) and the control line (C line) were located in the middle of the NC membrane and spaced 6 mm apart; after drying at 37°C for 12 h, the cellulose membrane was pasted on the middle part of the backing plate, the sample pad was overlapped with the T line end of the NC membrane by 1 mm, and the water absorption pad was pasted on the upper side of the cellulose membrane and overlapped with the cellulose membrane by 1 mm; the assembled test paper plate was cut into 3.5 mm wide test strips using a cutting machine.

[0098] 2. Preparation of gold-labeled antibody Colloidal gold suspension with an average diameter of 30 nm was prepared by reducing chloroauric acid with trisodium citrate, and gold-labeled antibody was prepared by using the colloidal gold to label the monoclone antibody of saffron prepared in Example 3, in the following manner: Take 1 mL of colloidal gold solution, adjust the pH to about 8.0 by adding 0.2 mol / L K2CO3 solution, add 10 μg of the monoclone antibody of saffron prepared in Example 3 and incubate for 30 min, then add 10 wt% BSA solution and incubate for 30 min, centrifuge at 10000 rpm for 20 min at 4°C, remove the supernatant, resuspend with 200 μL of 0.2 mol / L pH 7.4 phosphate buffer solution (containing 0.5% v / v / tween-20, 0.5% wt BSA, 5% wt sucrose, 0.3 wt% polyvinylpyrrolidone PVP and 0.03% v / v procline-300), and store at 4°C.

[0099] 3. Preparation of sample solution for detection Mix the sample to be detected with PBST in a volume ratio of 1:900 to obtain a sample solution.

[0100] 4. Detection procedure Take 120 μL of the sample solution and mix with 5 μL of the gold-labeled antibody by repeatedly pipetting, incubate at room temperature for 5 min, then insert the test strip into the sample solution and react for 3 min, then remove the test strip and the sample pad, and determine the result after 3-5 min.

[0101] 5. Determination of detection result Figure 5 is a test result determination chart for detecting saffron by using the colloidal gold test strip described above, as shown in Figure 5 If the sample does not contain the substance to be detected, saffron, the gold-labeled antibody is combined with the coated antigen on the T line (test line) of the rapid detection test strip, so that the test line shows a clear red line, indicating that the sample is negative (as shown in Figure 5 If the sample contains the substance to be detected, saffron, the saffron is combined with the gold-labeled antibody and cannot be captured by the test line of the rapid detection test strip, so that the test line does not develop color, indicating that it is positive (as shown in Figure 5 Similarly, the gold-labeled antibody is combined with the goat anti-mouse IgG on the C line (control line) of the cellulose membrane, so that the control line shows red color, indicating that the detection result of the test strip is valid (as shown in Figure 5 A and B), and if the control line has no color, it indicates that the detection result of the test strip is invalid (as shown in Figure 5 C and D).

[0102] In summary, the present invention provides two safrole haptens with spacer arms of appropriate lengths, which can fully expose the hapten after coupling with a carrier protein, and can avoid the recognition of the spacer arm as much as possible, so as to produce better antibodies to recognize safrole. The present invention also uses safrole haptens to prepare artificial antigens and antibodies, and the prepared safrole monoclonal antibodies have high titer, strong specificity, and high affinity. The immunoassay constructed using the safrole artificial antigens and antibodies provided by the present invention has high specificity and sensitivity, and can achieve rapid qualitative and quantitative detection of safrole in samples. The present invention simplifies the synthesis steps of the hapten, reduces the detection limit of the antibody, and improves the specificity and sensitivity of the safrole antibody.

[0103] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and are not intended to limit the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0104] Although the present invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions, and / or additions may be made and that substantial equivalents may be substituted for the elements of the embodiments without departing from the spirit and scope of the present invention. Additionally, many modifications may be made to adapt specific circumstances or materials to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the disclosed specific embodiments for carrying out the present invention, but rather to include all embodiments within the scope of the appended claims. Furthermore, unless specifically stated, any use of the terms first, second, etc. does not indicate any order or importance, but rather uses the terms first, second, etc. to distinguish one element from another.

Claims

1. A safrole hapten, characterized in that: Its structure is shown in formula (I) or formula (II): 。 2. The method for preparing the safrole hapten according to claim 1, characterized in that: include: allowing a first mixed reaction system containing eugenol, pyridine and aluminum halide to undergo a first reaction at 100-120° C. to obtain 4-allylcatechol; allowing a second mixed reaction system containing the 4-allylcatechol, methyl dichloroacetate, and an acid-binding agent to undergo a second reaction at 80-100° C. to obtain methyl 5-allylbenzo[D][1,3]dioxolane-2-carboxylate; The 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid methyl ester is hydrolyzed under alkaline conditions, and then the pH value of the hydrolysis reaction product is adjusted to acidic to obtain 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid, i.e., the safrole hapten shown in formula (II).

3. The preparation method according to claim 2, wherein: The molar equivalents of eugenol, pyridine and aluminum halide in the first mixed reaction system satisfy 1:1-3:1-2; And / or, the first reaction time is 10 to 12 hours; and / or, the molar equivalents of 4-allylcatechol, methyl dichloroacetate, and acid binding agent in the second mixed reaction system satisfy 1:1-3:1-3; And / or, the second reaction time is 6 to 8 hours; and / or, mixing the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid methyl ester and a base in a molar equivalent ratio of 1:1 to 3, and performing the hydrolysis at 0 to 40° C.; and / or, adjusting the pH value of the hydrolysis reaction product to 2-4.

4. The preparation method according to claim 2, characterized in that include: Mixing the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid and N,N'-carbonyldiimidazole, then adding tert-butyl aminobutyrate, and performing a fourth reaction at 0-40° C. to obtain tert-butyl (5-allylbenzo[D][1,3]dioxolane-2-carbonyl)aminobutyrate; The (5-allylbenzo[D][1,3]dioxolane-2-carbonyl)aminobutyric acid tert-butyl ester is mixed with an acid and subjected to a fifth reaction at 0-40° C. to obtain (5-allylbenzo[D][1,3]dioxolane-2-carbonyl)aminobutyric acid, i.e., the safrole hapten represented by formula (I).

5. The preparation method according to claim 4, characterized in that: The molar equivalents of 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid, N,N'-carbonyldiimidazole, and tert-butyl aminobutyrate satisfy 1:1-2:1-2; and / or, mixing the 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid and N,N'-carbonyldiimidazole for reaction for 0.5 to 2 hours, and then adding the tert-butyl aminobutyrate for reaction for 2 to 4 hours; and / or the molar equivalent of the tert-butyl (5-allylbenzo[D][1,3]dioxolane-2-carbonyl)aminobutyrate to the acid satisfies 1:1-3; And / or, the fifth reaction time is 4 to 6 hours.

6. A safrole artificial antigen, characterized in that: It is obtained by coupling the safrole hapten to the carrier protein according to claim 1, and the structure of the safrole artificial antigen is shown in formula (III) or formula (IV): ； Among them, Protein is a carrier protein.

7. The safrole artificial antigen according to claim 6, wherein: The carrier protein is any one of bovine serum albumin, chicken ovalbumin, hemocyanin and lactoferrin.

8. A safrole artificial antigen combination, characterized in that: The invention comprises an immunogen and a coating agent, wherein the immunogen is the artificial safrole antigen according to claim 6, and its structure is as shown in formula (III), and the carrier protein is bovine serum albumin; the coating agent is the artificial safrole antigen according to claim 6, and its structure is as shown in formula (IV), and the carrier protein is chicken ovalbumin.

9. A safrole antibody, characterized in that The method is obtained by immunizing an animal with the safrole artificial antigen as claimed in claim 6 or 7.

10. The safrole antibody according to claim 9, wherein: The safrole antibody is a monoclonal antibody; And / or, the safrole antibody is obtained by immunizing an animal with an artificial safrole antigen represented by formula (III) in claim 6 and whose carrier protein is bovine serum albumin.

11. Use of the safrole hapten according to claim 1, the safrole artificial antigen according to claim 6 or 7, the safrole artificial antigen combination according to claim 8, or the safrole antibody according to claim 9 in preparing a safrole detection product or in detecting safrole for purposes other than disease treatment and diagnosis.

12. A safrole detection device, characterized in that: The invention comprises the safrole artificial antigen according to claim 6 or 7 and the safrole antibody according to claim 9 or 10.

13. The safrole detection device according to claim 12, characterized in that: The safrole detection device includes a test strip assembly or an ELISA kit; And / or, the structure of the safrole artificial antigen is as shown in formula (IV) and the carrier protein is chicken ovalbumin, and the safrole antibody is obtained by immunizing an animal with a safrole artificial antigen having the structure shown in formula (III) and the carrier protein being bovine serum albumin; And / or, the safrole antibody is a monoclonal antibody.

14. An immunoassay method for detecting safrole, characterized in that: The detection is performed using the safrole artificial antigen according to claim 6 or 7 and the safrole antibody according to claim 9 or 10.

15. The immunoassay method according to claim 14, wherein: The structure of the safrole artificial antigen is as shown in formula (IV) and the carrier protein is chicken ovalbumin, and the safrole antibody is obtained by immunizing an animal with the safrole artificial antigen having the structure as shown in formula (III) and the carrier protein being bovine serum albumin; and / or, the safrole antibody is a monoclonal antibody; And / or, the immunoassay method is colloidal gold immunochromatography or ELISA.

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