A safrole hapten and its preparation method, artificial antigen, antibody and application

Abstract

This invention provides a safrole hapten, its preparation method, artificial antigen, antibody, and applications. The structure of the safrole hapten is shown below. The safrole hapten provided by this invention has a spacer arm of suitable length. After coupling with a carrier protein, the hapten is fully exposed while avoiding recognition of the spacer arm, thus generating a more effective antibody for recognizing safrole. This invention uses the safrole hapten to prepare artificial antigens and antibodies, obtaining safrole monoclonal antibodies with high titers, strong specificity, and high affinity. The limit of detection (LOD) of the ELISA detection method constructed based on the safrole artificial antigen and antibody of this invention is 2.50 ng / mL, and the half-inhibition concentration (IC50) is [missing value]. 50 The concentration was 20.04 ng / mL, and the quantitative detection range was 5.39–74.60 ng / mL. It showed no cross-reactivity with safrole analogues and could be used for rapid qualitative and quantitative detection of safrole in samples.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a safrole hapten and its preparation method, artificial antigen, antibody and application. Background Technology

[0002] Safrole (4-allyl-1,2-methylenedioxybenzene, C 10 H 10 Safrole (O2) is a naturally occurring organic compound belonging to the phenylpropene class of compounds. It is widely distributed in plants of the Lauraceae family (such as sassafras and camphor trees) and the Piperaceae family. In rotten and deteriorated spice plants, the content of safrole increases dramatically.

[0003] Safrole is toxic, can damage the liver, and may be carcinogenic. Currently, common methods for safrole detection include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). These methods suffer from complex and cumbersome sample preparation, expensive equipment, and high costs, and cannot achieve efficient and rapid detection and monitoring of safrole. Furthermore, the detection limit of the currently reported safrole immunoassay method is only 200 ng / mL, indicating low sensitivity. Therefore, to strengthen the regulation of safrole, there is an urgent need to establish a rapid detection method for safrole that is simple to operate, highly sensitive, and accurate. Summary of the Invention

[0004] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions:

[0005] A first aspect of the present invention provides a safrole hapten, the structure of which is shown in formula (I) or formula (II):

[0006] .

[0007] The two safrole haptens provided by this invention have spacer arms of suitable length. After coupling with carrier proteins, both allow the haptens to be fully exposed while minimizing recognition of the spacer arm, thus generating superior antibody recognition of safrole. If the spacer arm of the hapten is too short, the carrier protein may mask the characteristic structure of the analyte, resulting in a poor antibody response. If the spacer arm is too long, the hapten molecule may fold conformationally, becoming masked by the carrier protein, and thus more readily generating antibodies targeting the spacer arm.

[0008] A second aspect of the present invention provides a method for preparing the safrole hapten, comprising:

[0009] The first mixed reaction system containing sesamol, bromopropene and acid-binding agent was subjected to a first reaction at 60~100℃ to obtain 5-(allyloxy)benzo[D][1,3]dioxolane;

[0010] The 5-(allyloxy)benzo[D][1,3]dioxolane was heated to 180-200°C to carry out a second reaction to obtain 6-allylbenzo[d][1,3]dioxolane-5-ol;

[0011] The second mixed reaction system containing 6-allylbenzo[d][1,3]dioxolane-5-ol, an acid-binding agent, and tert-butyl bromide carboxylate was subjected to a third reaction at 60-100°C to obtain the intermediate.

[0012] The third mixed reaction system containing the intermediate and acid is subjected to a fourth reaction at 0~40°C to obtain the safrole hapten;

[0013] The tert-butyl bromocarboxylic acid ester is either tert-butyl 4-bromobutyrate or tert-butyl 2-bromoacetic acid.

[0014] When the tert-butyl bromocarboxylic acid is tert-butyl 4-bromobutyrate, the structure of the safrole hapten obtained is shown in formula (I); when the tert-butyl bromoalkylcarboxylic acid is tert-butyl 2-bromoacetic acid, the structure of the safrole hapten obtained is shown in formula (II).

[0015] In some embodiments, the molar equivalents of sesamol, bromopropene, and the acid-binding agent in the first mixed reaction system satisfy 1:(1~2):(1~2). The acid-binding agent can be any base known in the art that is commonly used as an acid-binding agent, such as, but not limited to, potassium carbonate.

[0016] In some embodiments, the first mixed reaction system further includes a first solvent, which may be any aprotic solvent known in the art, such as acetonitrile, but is not limited thereto.

[0017] In some embodiments, the first reaction takes 3 to 6 hours.

[0018] In some embodiments, the second reaction takes 3 to 6 hours.

[0019] In some embodiments, the molar equivalents of 6-allylbenzo[d][1,3]dioxolane-5-ol, tert-butyl bromide, and the acid-binding agent in the second mixed reaction system satisfy 1:(1~2):(1~2). The acid-binding agent can be any base known in the art that is commonly used as an acid-binding agent, such as, but not limited to, potassium carbonate.

[0020] In some embodiments, the second mixed reaction system further includes a second solvent, which may be any aprotic solvent known in the art, such as acetonitrile, but is not limited thereto.

[0021] In some embodiments, the third reaction takes 3 to 6 hours.

[0022] In some embodiments, the molar equivalent of the intermediate and the acid in the third mixed reaction system is 1:1 to 3. The acid may be, for example, hydrochloric acid, but is not limited thereto.

[0023] In some embodiments, the third mixed reaction system further includes a solvent, which includes, but is not limited to, 1,4-dioxane and / or tetrahydrofuran.

[0024] In some embodiments, the fourth reaction takes 3 to 6 hours.

[0025] A third aspect of the present invention provides a safrole artificial antigen, which is obtained by conjugating a carrier protein with the safrole hapten described in any of the above-mentioned technical solutions, wherein the structure of the safrole artificial antigen is shown in formula (III) or formula (IV):

[0026] ;

[0027] Protein is a carrier protein.

[0028] The safrole artificial antigen with the structure shown in formula (III) is obtained by conjugating a carrier protein to the safrole hapten with the structure shown in formula (I). For example, the carrier protein can be conjugated via the active ester method.

[0029] The safrole artificial antigen with the structure shown in formula (IV) is obtained by conjugating a carrier protein to the safrole hapten with the structure shown in formula (II). For example, the carrier protein can be conjugated via an active ester method.

[0030] In some embodiments, the carrier protein is any one of bovine serum albumin, chicken ovalbumin, hemocyanin, and lactoferrin, but is not limited thereto.

[0031] In some embodiments, the structure of the safrole artificial antigen is shown in formula (Ⅲ) and the carrier protein is bovine serum albumin.

[0032] In some embodiments, the structure of the safrole artificial antigen is shown in formula (Ⅳ) and the carrier protein is chicken ovalbumin.

[0033] A fourth aspect of the present invention provides a safrole artificial antigen combination, comprising an immunogen and a coating antigen, wherein the immunogen is a safrole artificial antigen as shown in Formula (III) with bovine serum albumin as the carrier protein, and the coating antigen is a safrole artificial antigen as shown in Formula (IV) with chicken ovalbumin as the carrier protein. The safrole antibody prepared based on this combination not only specifically recognizes the target analyte safrole, but also exhibits superior antibody sensitivity.

[0034] The fifth aspect of the present invention provides a safrole antibody, which is obtained by immunizing animals with the safrole artificial antigen described in any of the technical solutions.

[0035] In some embodiments, the safrole antibody is a monoclonal antibody.

[0036] In some embodiments, the safrole antibody is obtained by immunizing animals with a safrole artificial antigen of formula (III) and a carrier protein of bovine serum albumin.

[0037] The sixth aspect of the present invention provides the use of the safrole hapten, the artificial safrole antigen, the combination of the artificial safrole antigens, or the safrole antibody described in any of the technical solutions in the preparation of safrole detection products or in the detection of safrole for non-disease treatment diagnosis purposes.

[0038] A seventh aspect of the present invention provides a safrole detection device, comprising the safrole artificial antigen as described in any one of the technical solutions, and the safrole antibody as described in any one of the technical solutions.

[0039] 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 animals with the safrole artificial antigen as shown in formula (III) and the carrier protein is bovine serum albumin.

[0040] In some embodiments, the safrole antibody is a monoclonal antibody.

[0041] In some embodiments, the safrole detection device is a reagent kit.

[0042] In some embodiments, the safrole detection device is an ELISA kit.

[0043] In some embodiments, the ELISA kit further includes an enzyme-labeled plate, safrole standard, and substrate chromogenic solution, wherein the safrole artificial antigen is coated on the enzyme-labeled plate.

[0044] In some embodiments, the substrate developing solution contains urea peroxide and tetramethylbenzidine.

[0045] In some embodiments, the ELISA kit further includes a stop solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, and an enzyme-labeled secondary antibody dilution solution.

[0046] In some embodiments, the terminating solution comprises 8% to 12% H2SO4 by volume. In some preferred embodiments, the terminating solution comprises 10% H2SO4 by volume.

[0047] In some embodiments, the washing solution comprises 0.5%–1.0% (v / v) Tween-20, 0.01%–0.03% (w / w) sodium azide preservative, and 0.1 mol / L–0.3 mol / L phosphate buffer, and the pH of the washing solution is 7.2–7.6. In some preferred embodiments, the washing solution comprises 0.8% (v / v) Tween-20, 0.02% (w / w) sodium azide preservative, and 0.2 mol / L phosphate buffer, and the pH of the washing solution is 7.4.

[0048] In some embodiments, the blocking solution comprises 1%–3% casein and 0.1 mol / L–0.3 mol / L phosphate buffer, and the pH of the blocking solution is 7.1–7.5. In some preferred embodiments, the blocking solution comprises 2% casein and 0.2 mol / L phosphate buffer, and the pH of the blocking solution is 7.3.

[0049] In some embodiments, the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody.

[0050] In some embodiments, the enzyme-labeled secondary antibody diluent is a 0.1 mol / L to 0.3 mol / L phosphate buffer. In some preferred embodiments, the enzyme-labeled secondary antibody diluent includes a 0.2 mol / L phosphate buffer.

[0051] The substrate chromogenic solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody diluent in the ELISA kit can all be any known substances or formulations in the art. The components of the substrate chromogenic solution, stop solution, washing solution, blocking solution, enzyme-labeled secondary antibody, and enzyme-labeled secondary antibody diluent described above are merely exemplary and are not intended to limit the present invention.

[0052] The eighth aspect of the present invention provides an immunoassay method for detecting safrole, wherein the immunoassay method uses the artificial safrole antigen and the safrole antibody as described in any of the technical solutions for detection. The immunoassay method is for non-disease treatment diagnosis purposes.

[0053] In some embodiments, the structure of the safrole artificial antigen is as shown in formula (IV), and the carrier protein is chicken ovalbumin. The safrole antibody is obtained by immunizing animals with the safrole artificial antigen shown in formula (III), and the carrier protein is bovine serum albumin. The specificity and sensitivity of the immunoassay method constructed based on this combination are further improved.

[0054] In some embodiments, the safrole antibody is a monoclonal antibody.

[0055] In some embodiments, the immunoassay detection method is ELISA. It can be indirect ELISA and / or competitive ELISA; in some embodiments, indirect competitive ELISA is preferred.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] (1) The two safrole haptens provided by the present invention have spacer arms of suitable length. After being coupled with the carrier protein, the haptens can be fully exposed, while the recognition of the spacer arms can be avoided as much as possible, thereby generating better antibodies to recognize safrole.

[0058] (2) Based on the above-mentioned hapten with a suitable length of spacer arm, the present invention prepares artificial antigen and antibody of safrole. The prepared safrole monoclonal antibody has high titer, strong specificity and high affinity, providing core raw materials for establishing specific detection of safrole.

[0059] (3) The immunoassay detection method constructed based on the above-mentioned artificial antigen and antibody of safrole has high sensitivity and strong specificity, with a limit of detection (LOD) of 2.50 ng / mL and a half-inhibition concentration (IC50) of 1 / 2. 50 The concentration is 20.04 ng / mL, and the quantitative detection range is 5.39~74.60 ng / mL. It has no cross-reactivity with safrole analogues and can perform rapid qualitative and quantitative detection of safrole in samples. The operation is simple and the detection results are accurate and reliable. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0061] Figure 1 This is a UV full-wavelength scan identification image of the safrole hapten SF-1, safrole artificial antigen SF-1-BSA and BSA prepared in the embodiments of the present invention.

[0062] Figure 2 This is a UV full-wavelength scan identification image of the safrole hapten SF-2, safrole artificial antigen SF-2-OVA and OVA prepared in the embodiments of the present invention.

[0063] Figure 3 This is a standard curve of the indirect competitive ELISA method constructed based on SF-2-OVA and safrole monoclonal antibody in the embodiments of the present invention. Detailed Implementation

[0064] The technical solutions of the present invention will be described in detail below with reference to 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 construed as limiting, but are merely intended to form the basis of the claims and to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0065] In addition, unless otherwise specified, all raw materials used in the following embodiments can be purchased from the market or other sources, and all production and testing equipment used are known in the art, as are the testing methods used.

[0066] Unless otherwise specified, in the following embodiments of the present invention, the amount of raw materials used in the form of "equivalent" refers to molar equivalent.

[0067] Example 1

[0068] This embodiment provides a safrole hapten and its synthesis method, and identifies the synthesized safrole hapten.

[0069] 1. Synthesis of safrole hapten SF-1

[0070] The synthetic route for the safrole hapten SF-1 is shown below:

[0071]

[0072] Specifically, the steps include the following:

[0073] (1) Dissolve 1 eq sesamol, 1.2 eq bromopropene and 1.2 eq potassium carbonate in acetonitrile and reflux overnight; after the reaction, extract with ethyl acetate and water and retain the organic phase; add 15 g silica gel powder to the organic phase and evaporate to dryness using a rotary evaporator. Purify by column chromatography using the developing solvent (volume ratio, petroleum ether: ethyl acetate: formic acid: 80:20:1) to obtain 5-(allyloxy)benzo[D][1,3]dioxolane.

[0074] (2) 5-(allyloxy)benzo[D][1,3]dioxolane was heated to 180 °C and reacted overnight. After the reaction was completed, it was dissolved in ethyl acetate. 15 g of silica gel powder was added to the organic phase and the mixture was evaporated to dryness using a rotary evaporator. The mixture was purified by column chromatography using the developing solvent (volume ratio: petroleum ether: ethyl acetate: formic acid: 79:30:1) to obtain 6-allylbenzo[d][1,3]dioxolane-5-ol.

[0075] (3) 1 eq of 6-allylbenzo[d][1,3]dioxolane-5-ol, 1.2 eq of potassium carbonate, and 1.2 eq of tert-butyl 4-bromobutyrate were added to acetonitrile and refluxed overnight. After the reaction was completed, the organic phase was extracted with ethyl acetate and water and retained. 15 g of silica gel powder was added to the organic phase and the mixture was evaporated to dryness using a rotary evaporator. The product was purified by column chromatography using the developing solvent (volume ratio: petroleum ether: ethyl acetate: 80:30) to obtain tert-butyl 6-(6-allylbenzo[D][1,3]dioxolane-5-oxy)butyrate.

[0076] (4) 1 eq of tert-butyl 6-(6-allylbenzo[D][1,3]dioxolane-5-oxy)butyrate and 3 eq of hydrochloric acid were added to 1,4-dioxane and reacted for 3 h. After the reaction was completed, the mixture was dried by rotary evaporator to obtain 4-(4-allylbenzo[D][1,3]dioxolane-5-oxy)butyrate, which is the safrole hapten SF-1.

[0077] 2. Identification of safrole hapten SF-1

[0078] The 1H NMR spectrum of the safrole hapten SF-1 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 11.83(s, 1H), 6.98 – 6.38 (m, 2H), 6.08 – 5.73 (m, 3H), 5.27 – 4.71 (m, 2H), 4.06(t, J = 6.2 Hz, 2H), 3.49 – 3.03 (m, 2H), 2.51 (t, J = 9.3 Hz, 2H), 2.08 (tt, J = 9.0, 6.2 Hz, 2H).

[0079] The mass spectrometry results for the safrole hapten were: MS: 264.1, ESI-[MH] + :263.1.

[0080] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the safrole hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the safrole hapten skeleton structure. This indicates that the safrole hapten, denoted as SF-1, was successfully prepared, and its structural formula is shown in formula (I).

[0081] .

[0082] 3. Synthesis of safrole hapten SF-2

[0083] The synthetic route for the safrole hapten SF-2 is shown below:

[0084]

[0085] The preparation method of safrole hapten SF-2 is basically the same as that of SF-1, except that tert-butyl 4-bromobutyrate is replaced with tert-butyl 2-bromoacetate. The rest is the same as the preparation method of SF-1, and will not be described in detail here.

[0086] 4. Identification of safrole hapten SF-2

[0087] The 1H NMR spectrum of the safrole hapten SF-2 is as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 12.90 (s, 1H), 6.66 (d, J = 3.8 Hz, 2H), 6.11 – 5.78 (m, 3H), 5.24 – 4.92 (m, 2H), 4.69 (s, 2H), 3.55 – 3.12 (m, 2H).

[0088] The mass spectrometry results for the safrole hapten were: MS: 236.07, ESI-[MH] + : 235.07.

[0089] The mass spectrometry and NMR results show that the mass spectrometry results correspond to the molecular weight of the safrole hapten, and the number of protons in the NMR spectrum corresponds to the number of protons in the safrole hapten skeleton structure. This indicates that the safrole hapten, denoted as SF-2, was successfully prepared, and its structural formula is shown in formula (II).

[0090] .

[0091] Example 2

[0092] This embodiment provides a safrole artificial antigen and its synthesis method, and identifies the synthesized safrole artificial antigen.

[0093] 1. Synthesis of safrole artificial antigen

[0094] (1) The safrole hapten SF-1 (3.156 mg, 0.012 mol) prepared in Example 1 was added to a brown reaction vial, 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 the hapten activation solution;

[0095] (2) Dissolve BSA (bovine serum albumin, 10 mg, 0.00015 mol) in 1 mL PBS buffer (0.01 mol / L, pH=7.4) to obtain BSA solution. Stir the BSA solution and hapten activation solution at 4 °C overnight.

[0096] (3) Collect the reaction solution, transfer it to the prepared dialysis bag, and then put it into 5 L of 0.01 M PBS buffer; dialyze at 4 ℃ for 3 days, and change the solution with pre-cooled 0.01 M PBS buffer every 8 hours;

[0097] (4) After dialysis purification, safrole artificial antigen, denoted as SF-1-BSA, was obtained. After concentration determination and ultraviolet spectroscopy identification, it was stored in a -20 ℃ refrigerator.

[0098] The artificial antigen SF-2-OVA prepared according to the above method is different from the preparation of SF-1-BSA only in that OVA (chicken egg albumin) is used instead of BSA, and SF-2 prepared in Example 1 is used instead of SF-1. The rest is the same as the preparation method of SF-1-BSA, and will not be repeated here.

[0099] 2. Identification of safrole artificial antigen

[0100] BSA, OVA, SF-1, SF-2, SF-1-BSA, and SF-2-OVA were identified by scanning using the ultraviolet full-wavelength method (200-350 nm).

[0101] Figure 1 These are ultraviolet full-wavelength scan images of safrole hapten SF-1, safrole artificial antigen SF-1-BSA, and BSA, as shown. Figure 1 As shown, by comparing the highest absorbance values ​​of each substance before and after conjugation, it was found that the absorption curve of SF-1-BSA is significantly different from that of the carrier protein BSA. SF-1 has a strong absorption peak above 343 nm, while after conjugation with BSA, the absorption peak of SF-1-BSA is similar to that of BSA at 230 nm but significantly higher than that of BSA at 280 nm, and the curve relative to the hapten SF-1 shows a significant shift. Since all unreacted components were removed by dialysis during the post-conjugation dialysis process, the characteristic peaks of the conjugation product are contributed by the protein-bound drug molecules, indicating that the reaction product is a complex of the carrier protein BSA and SF-1.

[0102] Figure 2 These are ultraviolet full-wavelength scan images for the identification of safrole hapten SF-2, safrole artificial antigen SF-2-OVA, and OVA, as shown below. Figure 2As shown, similarly, the absorption curve of SF-2-OVA showed characteristic peaks that were different from those of OVA and SF-2, indicating that the reaction product was a complex of the carrier protein OVA and SF-2.

[0103] The above results demonstrate that the artificial antigens SF-1-BSA and SF-2-OVA of safrole were successfully prepared in this invention, and their structural formulas are shown in formulas (III) and (IV), respectively:

[0104] ;

[0105] In formula (III), the carrier protein is BSA, and in formula (IV), the carrier protein is OVA.

[0106] Example 3

[0107] This embodiment provides a safrole monoclonal antibody and its preparation method, specifically including the following steps:

[0108] 1. Animal immunization

[0109] Using the safrole artificial antigen SF-1-BSA prepared in Example 2 as the immunogen, SF-1-BSA was diluted to 5 mg / mL with 0.01 mol / L PBS, then mixed with an equal volume of Freund's complete adjuvant and thoroughly emulsified. This mixture was then used to immunize 8-week-old female BALB / c mice. For the first immunization, three female BALB / c mice were subcutaneously inoculated at multiple sites in the abdomen, with an antigen dose of 100 μg / mouse, administered in 0.1 ml per mouse. A second immunization was performed 14 days later, using an equal volume of Freund's incomplete adjuvant emulsified with the immunogen, at the same dose as the first immunization. Three booster immunizations were administered. After immunization, tail serum was collected. Its titer and inhibition rate were determined. Mice with the best results were selected for a pulse immunization, with an antigen dose of 100 μg / mouse.

[0110] 2. Cell fusion

[0111] Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 1450) method, with the following specific steps:

[0112] a. After euthanizing mice by blood collection from the eyeballs, immediately disinfect them in 75% alcohol for about 5 minutes. Aseptically remove the spleen of the mice, grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1000 rpm, 7 min), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0113] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in RPMI 1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The number of SP2 / 0 tumor cells should reach (1-4) × 10⁻⁶ cells before fusion. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected and suspended in RPMI-1640 basal culture medium for cell counting during fusion.

[0114] c. Mix the two cell lines, bring the volume to 15 mL, centrifuge (1000 rpm / min, 7 min), and discard the supernatant. Remove the centrifuge tube, gently shake at 60°C in a circular motion, and then begin preparation for fusion. The fusion time should be controlled as follows:

[0115] First minute: Slowly add PEG dropwise;

[0116] Second minute: Continue rotating (1 minute);

[0117] Third minute: Add 1 mL of basal culture medium;

[0118] Fourth minute: Add 3 mL of basal culture medium;

[0119] Fifth minute: Add 8 mL of basal culture medium;

[0120] Sixth minute: Add 8 mL of basal culture medium;

[0121] After fusion, the cell culture was centrifuged (1000 rpm / min, 7 min), and the 200 mL HAT culture medium was removed and placed in a clean bench for later use.

[0122] d. After centrifuging the hybridoma cells, discard the supernatant, aspirate 2 mL of HAT medium into a centrifuge tube, gently and slowly pipette to mix evenly, then transfer the cell solution in the centrifuge tube to 200 mL of HAT medium, gently shake the medium to disperse the cells evenly, and then plate them.

[0123] 3. Cell selection and cell line establishment

[0124] On day 3 of cell fusion, the fused cells were screened with RPMI-1640 medium with a half-replacement. On day 5, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 2% 50×HT. On day 7, the cell supernatant was collected for screening.

[0125] The screening process consisted of two steps: First, positive cell wells were selected using ic-ELISA. Second, safrole was used as a standard, and its inhibitory effect on positive cells was determined using ic-ELISA. Cell wells that showed good inhibition of the safrole standard were selected, and subcloning was performed using the limiting dilution method. The cells were then tested using the same method after seven days. Subcloning was performed three times using the above method to finally obtain the safrole monoclonal antibody cell line AP-H6C.

[0126] 4. Preparation and identification of safrole monoclonal antibodies

[0127] BALB / c mice aged 8-10 weeks were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Ascites fluid was collected from safrole hybridoma cells starting on day 7. The ascites fluid was then purified for antibody treatment using immunoaffinity chromatography. Under neutral conditions, Protein G bound to IgG immunoglobulins in the ascites fluid, causing other impurities to flow out and be discarded. Then, IgG was desorbed from Protein G under acidic conditions using 0.01 M glycine buffer to obtain high-purity IgG immunoglobulins. The pH of the IgG immunoglobulin solution was adjusted to neutral using 0.01 M Tris-HCl buffer, followed by dialysis to desalt, ultimately yielding high-purity safrole monoclonal antibodies. The purified monoclonal antibodies were stored at -20 °C.

[0128] 5. Sensitivity evaluation of safrole monoclonal antibody (indirect competitive ELISA method)

[0129] Using the safrole artificial antigen SF-2-OVA prepared in Example 2 as the coating agent, it was diluted with coating buffer (0.05 M carbonate buffer, pH 9.6) to a concentration of 500 ng / mL, and 100 μL / well was used to coat a 96-well microplate. The plate was incubated overnight (12 h) at 4 °C.

[0130] Discard the coating solution, wash twice with PBST (0.01 M PBS, 0.06 % v / v Tween-20), and pat dry;

[0131] Add 120 μL of blocking buffer (PBST containing 7% skim milk powder) to each well and block at 37 °C for 1 h;

[0132] Discard the sealing liquid, tap the plate, dry at 37°C, and then pack it in a self-sealing bag for later use.

[0133] The safrole monoclonal antibody prepared using SF-1-BSA as an immunogen was diluted with PBST at a volume ratio of 1:128000. The safrole standard was diluted with PBST to different concentrations to obtain safrole standard dilutions with concentrations of 10000 ng / mL, 1000 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.

[0134] Add 50 μL / well of safrole standard dilution buffer at different concentrations (three parallel groups), then add 50 μL / well of diluted safrole monoclonal antibody, incubate at 37 ℃ for 40 min, wash five times, and pat dry;

[0135] Add 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash five times, and pat dry;

[0136] Add 100 μL / well of colorimetric reagent and develop for 10 min;

[0137] The reaction was terminated by adding 50 μL of 10% H2SO4 solution, and the OD value was read at 450 nm.

[0138] ELISA standard curve plotting: Plot B / B0 as the ordinate (B represents the absorbance OD of standards with different concentrations of safrole). 450 B0 is the absorbance value (OD) of the blank control well. 450 The standard curve was obtained by fitting the logarithm of the concentration of safrole standard to the x-axis using the Logistic function, and the formula for the standard curve was derived.

[0139] Figure 3 The standard curve of the indirect competitive ELISA method, constructed using SF-2-OVA as the coating antigen and the safrole monoclonal antibody prepared in this example, is shown below. Figure 3 As shown, the limit of detection (LOD) was 2.50 ng / mL, and the half-maximum inhibitory concentration (IC50) was [missing value]. 50 The concentration was 20.04 ng / mL, and the quantitative detection range was 5.39~74.60 ng / mL.

[0140] 6. Specificity evaluation of safrole monoclonal antibodies

[0141] Dihydrosafrole, piperine ring, and sesamol are safrole analogs. The specificity of the safrole monoclonal antibody prepared in this example was evaluated by cross-reactivity experiments.

[0142] The method for sensitivity evaluation described above differs only in that the safrole standard is replaced with standards for dihydrosafrole, piperine, and sesamol, and the IC50 values ​​of each structural analog are obtained by performing the tests at the same dilution factor.

[0143] The cross-reactivity rate (CR) of safrole is calculated using the following formula: CR (%) = IC50 (safrole) / IC50 (structural analog) × 100%. The lower the cross-reactivity rate, the stronger the specificity.

[0144] Table 1. Cross-reactivity results of safrole monoclonal antibodies with safrole and its analogues

[0145] ;

[0146] Note: NR indicates no reaction, meaning that the safrole monoclonal antibody of the present invention does not recognize this analogue.

[0147] As shown in Table 1, the cross-reactivity of the safrole monoclonal antibody to safrole was 100%, and the IC50 was [missing value]. 50 The concentration was 20.44 ng / mL, with no cross-contamination against dihydrosafrole, piperine, and sesamol. This indicates that the antibody used for detecting safrole has high recognition ability and specificity for safrole, and can effectively eliminate interference from methylenedioxybenzene analogs (such as dihydrosafrole, piperine, and sesamol), making it specifically suitable for the detection of safrole.

[0148] The above results indicate that the safrole monoclonal antibody prepared in this invention exhibits excellent detection performance, high sensitivity, and strong specificity for safrole.

[0149] Example 4

[0150] This embodiment provides a combination of safrole immunogen and coating antigen.

[0151] Using the safrole artificial antigen SF-2-OVA prepared in Example 2 as the coating antigen, and the safrole monoclonal antibody prepared in Example 3 using SF-1-BSA as the immunogen, the combined effect of the immunogen and coating antigen was evaluated by serum titers and inhibition rates obtained through an indirect competitive ELISA method. The specific operating steps are as follows:

[0152] 1. Dilute the coating agent to a concentration of 1000 ng / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), add 100 μL / well to coat a 96-well microplate, incubate overnight at 4 ℃, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0153] 2. Add 120 μL of blocking solution (PBST containing 7% skim milk powder) to each well, block at 37 ℃ for 1 h, discard the blocking solution, plate, and dry in a drying oven at 37 ℃ for later use;

[0154] 3. Dilute the safrole monoclonal antibody with PBST at volume ratios of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, and set up blank control wells (using PBST instead); dilute 1 mg / mL safrole standard 1000 times with PBST to obtain a safrole standard dilution solution with a concentration of 1 μg / mL;

[0155] 4. Potency column setup: First, add 50 μL of PBST to each well, then add 50 μL of safrole monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of the antibody.

[0156] 5. Inhibition column setup: First, add 50 μL of safrole standard dilution buffer to each well, then add 50 μL of safrole monoclonal antibody at different dilution ratios to each well, and finally add 50 μL of PBST to the last well instead of antibody.

[0157] 6. Incubate at 37℃ for 40 minutes, wash 5 times, and then plate.

[0158] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash 5 times, and plate.

[0159] 8. Add the color developing solution and incubate at 37 ℃ for 10 min;

[0160] 9. Terminate the reaction by adding 10% v / v H2SO4 and read the OD value at 450 nm; calculate the titer and inhibition rate. The titer is the OD value. 450 The antibody dilution factor corresponding to approximately 1.0 is: inhibition rate = (OD value of titer - OD value of inhibition) / OD value of inhibition × 100%.

[0161] This invention also employs the same methods described above to conjugate SF-1 with lactoferrin (LF), SF-1 with chicken ovalbumin, and SF-2 with lactoferrin to prepare artificial antigens SF-2-LF, SF-1-OVA, and SF-1-LF. Different combinations of immunogens and coating antigens were then constructed using these combinations, and the combined effects of the immunogens and coating antigens were evaluated using the same methods described above. The results are shown in Table 2.

[0162] Table 2. ELISA detection results for different immunogens and coating antigens.

[0163] ;

[0164] As shown in Table 2, the monoclonal antibody prepared using the artificial antigen SF-1-BSA of safrole as an immunogen and SF-2-OVA is the optimal combination of immunogen and coating agent. Under this combination, the monoclonal antibody can not only specifically recognize the target analyte safrole, but also has good antibody sensitivity and can be used to specifically recognize the target analyte safrole.

[0165] Example 5

[0166] This embodiment provides an ELISA kit for detecting safrole, specifically including:

[0167] 1. Composition

[0168] (1) The enzyme-labeled plate coated with the coating agent is prepared by the following method:

[0169] The SF-2-OVA prepared in Example 2 was used as the coating agent. It was diluted to 500 ng / mL with the coating stock solution (0.05 M carbonate buffer, pH 9.6). 100 μL of the solution was added to coat 96-well microplates and incubated overnight at 4°C in the dark. The liquid in the wells was discarded, and the plates were washed twice with the washing solution in this kit for 30 s each time. The plates were then patted dry. Then, the blocking solution in this kit was added at 120 μL / well and incubated at 37°C in the dark for 1 h. The liquid in the wells was discarded, and the plates were patted dry. After drying, the plates were vacuum sealed with aluminum foil for storage.

[0170] (2) Standards: 12 different concentrations of safrole standards, and safrole standard dilutions at concentrations of 10000 ng / mL, 1000 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;

[0171] (3) Antibody: Safrole monoclonal antibody prepared in Example 3;

[0172] (4) Enzyme-labeled secondary antibody: Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0173] (5) Substrate colorimetric solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0174] (6) Termination solution: 2 mol / L H2SO4;

[0175] (7) Washing solution: pH value is 7.4, containing 0.8% Tween-20 by volume, 0.02% sodium azide preservative by mass, and 0.2 mol / L phosphate buffer; dilute the washing solution 20 times with water before use (i.e., add 1 part washing solution to 19 parts water, prepare fresh before use) to obtain the washing solution working solution;

[0176] (8) Diluent: 0.2 mol / L phosphate buffer; dilute the diluent 20 times with water before use (i.e., add 1 part of diluent to 19 parts of water, prepare fresh before use) to obtain the working solution of the diluent;

[0177] (9) Blocking solution: pH 7.3, 0.2 mol / L phosphate buffer containing 2% casein.

[0178] 2. Instructions for use

[0179] (1) Sample testing

[0180] Number the wells corresponding to the samples and standards in this kit sequentially. Perform two parallel wells for each sample and standard, and record the positions of the standard and sample wells. Dilute the antibody with the required amount of diluent at a 1:40 volume ratio (i.e., add 1 part antibody to 40 parts diluent; prepare fresh before use) to obtain the antibody working solution. Dilute the enzyme-labeled secondary antibody with the required amount of diluent at a 1:10 volume ratio (i.e., add 1 part enzyme-labeled secondary antibody to 10 parts diluent; prepare fresh before use) to obtain the enzyme-labeled secondary antibody working solution.

[0181] Add 50 μL of standard or sample to the corresponding well, then add 50 μL of antibody working solution to the corresponding well, gently shake to mix, cover with a cover plate and incubate at 25 ℃ in the dark for 40 min.

[0182] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10 s intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0183] Add 100 μL / well of enzyme-labeled secondary antibody working solution to the corresponding microwell, gently shake to mix, cover with a cover plate membrane, and incubate at 25 ℃ in the dark for 30 min.

[0184] Shake off the liquid in the well and add 250 μL of washing working solution per well. Wash thoroughly 4-5 times, with 10 s intervals between each wash. Discard the washing working solution in the well and pat dry with absorbent paper (any air bubbles not removed after patting can be punctured with an unused pipette tip).

[0185] Add 50 μL of substrate chromogenic solution A per well, then add 50 μL of substrate chromogenic solution B per well, gently shake to mix, cover with a cover plate and incubate at 25 °C in the dark for 10 min.

[0186] Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0187] (2) Drawing the standard curve

[0188] Plot B / B0 as the ordinate (B represents the absorbance OD of standards at different concentrations). 450 B0 is the absorbance value (OD) of the blank control well. 450 The standard curve was obtained by using the logarithm of the concentration of the standard as the abscissa and performing curve fitting with the Logistic function.

[0189] (3) Calculation of sample concentration

[0190] OD of the sample 450 Substituting the average value into the formula of the standard curve above, we obtain the concentration of the sample. Multiplying this by the corresponding dilution factor gives the actual concentration of safrole in the sample.

[0191] In summary, the artificial antigens prepared using the two safrole haptens provided in this invention, due to the appropriately long spacer arms connecting the target epitope and the carrier protein, not only keep the characteristic structure of the target molecule away from the carrier protein but also maximize the exposure of the hapten to the immune system. Based on this, the resulting safrole monoclonal antibodies exhibit high titer, strong specificity, and high affinity. The ELISA detection method constructed based on the safrole artificial antigens and monoclonal antibodies provided in this invention demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 2.50 ng / mL and a half-inhibitory concentration (IC50). 50 The concentration is 20.04 ng / mL, and the quantitative detection range is 5.39~74.60 ng / mL. It has no cross-reactivity with safrole analogues and can perform rapid qualitative and quantitative detection of safrole in samples. The operation is simple and the detection results are accurate and reliable.

[0192] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0193] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0194] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A safrole artificial antigen combination, characterized in that, It includes an immunogen and a coating antigen, wherein the structure of the immunogen is shown in formula (III) and the carrier protein is bovine serum albumin, and the structure of the coating antigen is shown in formula (IV) and the carrier protein is chicken ovalbumin. 。 2. The use of the safrole artificial antigen combination according to claim 1 in the preparation of safrole detection products or in the detection of safrole for the purpose of non-disease treatment diagnosis.

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