A safinin hapten and its application in the detection of safinin in immunoassay

Abstract

The application provides a safinin hapten and application thereof in immunological analysis for detecting safinin. The structure of the safinin hapten is shown in the following formula: two safinin haptens provided by the application have a spacer arm with a proper length, which can make the hapten fully exposed after being coupled with a carrier protein and can avoid recognizing the spacer arm as much as possible. The safinin hapten is used to prepare artificial antigens and antibodies, and the safinin monoclonal antibody obtained has high titer, high specificity and high affinity. The immunological analysis method constructed based on the safinin artificial antigen and the antibody has high specificity and sensitivity, the minimum 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, and there is no cross reaction to the analogues of safinin, so that the safinin in a sample can be rapidly qualitatively and quantitatively detected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection, and particularly relates to a safrole hapten, a preparation method thereof, an artificial antigen, an antibody, and application of the antibody to immunological analysis and detection of safrole. BACKGROUND

[0002] Safrole (4-allyl-1,2-methylenedioxybenzene, C 10 H 10 O2) is a naturally occurring organic compound belonging to phenylpropenes, and is a volatile oil component of many plants. Safrole exists in a liquid form, has a sweet and pungent odor, and is widely distributed in plants of the Lauraceae (such as Cinnamomum camphora) and Piperaceae.

[0003] Safrole is toxic and can cause damage to the liver and may be carcinogenic. Animal experiments have shown that safrole can cause gene mutation and liver tumors. In the case of spoilage of spice plants, the content of safrole increases dramatically.

[0004] Currently, methods commonly used for detection of safrole include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). These methods have the problems of complex and tedious pretreatment, expensive instruments, and high cost, and cannot achieve efficient and rapid detection and monitoring of safrole. In addition, the reported immunological analysis method for safrole has a detection limit of only 200 ng / mL, and has low sensitivity. Therefore, it is necessary to establish a rapid detection method for safrole which is simple to operate, has high sensitivity, and is highly accurate. SUMMARY

[0005] To solve all or part of the above technical problems, the present application provides the following technical solutions:

[0006] The first aspect of the present application provides a safrole hapten, the structure of which is shown in formula (I) or formula (II):

[0007] .

[0008] The two safrole haptens provided by the present application have a spacer arm of appropriate length, which can fully expose the hapten after coupling with the carrier protein and can avoid recognition of the spacer arm as much as possible, thereby producing a more optimal antibody recognizing safrole. If the spacer arm of the hapten is short, the carrier protein will mask the characteristic structure of the analyte, thereby leading to poor antibody reaction. If the spacer arm is long, the conformation of the hapten molecule will fold, thereby being masked by the carrier protein, and an antibody against the spacer arm is easily produced. The safrole hapten having the above structure provided by the present application has a spacer arm of a certain length connected between the target epitope of the hapten and the carrier protein, thereby making the characteristic structure of the target molecule away from the carrier protein and maximizing the exposure of the hapten to the immune system.

[0009] The second aspect of the present application provides a preparation method of the sa- lonin hapten.

[0010] The preparation method of the sa- lonin hapten as shown in Formula (II) comprises:

[0011] carrying out a first reaction on a first mixed reaction system containing syringol, pyridine and aluminum halide at 100-120℃ to obtain 4-allyl pyrocatechol;

[0012] carrying out a second reaction on a second mixed reaction system containing the 4-allyl pyrocatechol, methyl dichloroacetate and an acid binding agent at 80-100℃ to obtain 5-allyl benzo[D][1,3]dioxolane-2-carboxylic acid methyl ester;

[0013] hydrolyzing the 5-allyl benzo[D][1,3]dioxolane-2-carboxylic acid methyl ester under alkaline conditions, and then adjusting the pH value of the hydrolyzed reaction product to be acidic to obtain 5-allyl benzo[D][1,3]dioxolane-2-carboxylic acid, i.e. the sa- lonin hapten as shown in Formula (II).

[0014] In some embodiments, the molar equivalent of syringol, pyridine and aluminum halide in the first mixed reaction system satisfies 1:1-3:1-2. The aluminum halide includes but is not limited to aluminum chloride.

[0015] In some embodiments, the time of the first reaction is 10-12h.

[0016] In some embodiments, the first mixed reaction system further comprises a solvent, which includes one or more combinations of acetonitrile, dimethylformamide and dimethyl sulfoxide, but is not limited thereto.

[0017] In some embodiments, the molar equivalent of 4-allyl pyrocatechol, methyl dichloroacetate and the acid binding agent in the second mixed reaction system satisfies 1:1-3:1-3. The acid binding agent includes but is not limited to potassium carbonate.

[0018] In some embodiments, the second mixed reaction system further comprises a solvent, which can be any known aprotic solvent in the art, for example, including but not limited to acetonitrile and the like.

[0019] In some embodiments, the time of the second reaction is 6-8h.

[0020] In some embodiments, the 5-allyl benzo[D][1,3]dioxolane-2-carboxylic acid methyl ester is mixed with a base according to a molar equivalent of 1:1-3, and the hydrolysis is carried out at 0-40℃.

[0021] In some embodiments, the hydrolysis is performed by adding the 5-allylbenzo[D][1,3]dioxol-2-carboxylic acid methyl ester and a base to an aqueous solution of 1,4-dioxane.

[0022] In some embodiments, the pH of the hydrolyzed reaction product is adjusted to 2-4.

[0023] The method for preparing the sahnin hapten of Formula (I) comprises:

[0024] The 5-allylbenzo[D][1,3]dioxol-2-carboxylic acid is mixed with N,N'-carbonyldiimidazole, and then tert-butyl aminobutyrate is added, and a fourth reaction is performed at 0-40°C to obtain tert-butyl (5-allylbenzo[D][1,3]dioxol-2-carbonyl) aminobutyrate.

[0025] The tert-butyl (5-allylbenzo[D][1,3]dioxol-2-carbonyl) aminobutyrate is mixed with an acid and a fifth reaction is performed at 0-40°C to obtain (5-allylbenzo[D][1,3]dioxol-2-carbonyl) aminobutyric acid, i.e., the sahnin hapten of Formula (I).

[0026] The method for preparing the 5-allylbenzo[D][1,3]dioxol-2-carboxylic acid is the same as described above.

[0027] In some embodiments, the molar equivalents of the 5-allylbenzo[D][1,3]dioxol-2-carboxylic acid, N,N'-carbonyldiimidazole, and tert-butyl aminobutyrate satisfy 1:1-2:1-2.

[0028] In some embodiments, the 5-allylbenzo[D][1,3]dioxol-2-carboxylic acid is first mixed with N,N'-carbonyldiimidazole for 0.5-2 h, and then the tert-butyl aminobutyrate is added and reacted for 2-4 h.

[0029] In some embodiments, the fourth reaction is performed in a solvent, and the solvent includes dichloromethane, but is not limited thereto.

[0030] In some embodiments, the molar equivalents of the tert-butyl (5-allylbenzo[D][1,3]dioxol-2-carbonyl) aminobutyrate and the acid satisfy 1:1-3.

[0031] In some embodiments, the fifth reaction is performed in a solvent, and the solvent includes 1,4-dioxane, but is not limited thereto.

[0032] In some embodiments, the time for the fifth reaction is 4-6 h.

[0033] The third aspect of the present application provides a safinin artificial antigen, which is obtained by coupling a carrier protein with the safinin hapten, and the structure of the safinin artificial antigen is shown in formula (III) or formula (IV).

[0034] ;

[0035] In the formula, Protein is a carrier protein.

[0036] The safinin artificial antigen with the structure shown in formula (III) is obtained by coupling a carrier protein with the safinin hapten with the structure shown in formula (I). For example, the coupling of the carrier protein is achieved by the active ester method.

[0037] The safinin artificial antigen with the structure shown in formula (IV) is obtained by coupling a carrier protein with the safinin hapten with the structure shown in formula (II). For example, the coupling of the carrier protein is achieved by the active ester method.

[0038] In some embodiments, the carrier protein is any one of bovine serum albumin, chicken egg albumin, hemocyanin, lactoferrin, but is not limited thereto.

[0039] In some embodiments, the structure of the safinin artificial antigen is shown in formula (III) and the carrier protein is bovine serum albumin.

[0040] In some embodiments, the structure of the safinin artificial antigen is shown in formula (IV) and the carrier protein is chicken egg albumin.

[0041] The fourth aspect of the present application provides a safinin artificial antigen combination, which comprises an immunogen and a coating agent, the immunogen is a safinin artificial antigen shown in formula (III) and the carrier protein is bovine serum albumin, and the coating agent is a safinin artificial antigen shown in formula (IV) and the carrier protein is chicken egg albumin.

[0042] The fifth aspect of the present application provides a safinin antibody, which is obtained by immunizing an animal with any one of the safinin artificial antigens.

[0043] In some embodiments, the safinin antibody is a monoclonal antibody.

[0044] In some embodiments, the safinin antibody is obtained by immunizing an animal with a safinin artificial antigen shown in formula (III) and the carrier protein is bovine serum albumin.

[0045] The sixth aspect of the present application provides the use of the safinin hapten of any one of the technical solutions, the safinin artificial antigen of any one of the technical solutions, the safinin artificial antigen combination, or the safinin antibody of 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.

[0046] The seventh aspect of the present application provides a safrole detection device, which comprises the safrole artificial antigen according to any one of the technical solutions and the safrole antibody according to any one of the technical solutions.

[0047] In some embodiments, the safrole detection device comprises a test strip assembly or an ELISA kit.

[0048] In some embodiments, the test strip assembly comprises a test strip and a reaction cup, the test strip contains a detection line and a control line, the detection line is coated with the safrole artificial antigen, and the control line is coated with a goat anti-mouse monoclonal antibody, and the reaction cup contains the colloidal gold-labeled safrole antibody.

[0049] In some embodiments, the test strip comprises a base plate, a sample pad, a cellulose membrane and a water absorption pad arranged on the base plate in sequence, and the cellulose membrane is provided with the detection line and the control line.

[0050] In detection, the sample to be detected is added into the reaction cup, mixed with the colloidal gold-labeled safrole antibody in the reaction cup, and then the mixture is added dropwise to the water absorption pad of the test strip, and the result is determined after the mixture is fully infiltrated into the water absorption pad.

[0051] The determination method is as follows: if the control line does not develop color, the detection result is invalid, and the detection needs to be re-performed; if the control line develops color, the detection result is valid, and the detection line is further determined: if the detection line does not develop color or develops color weakly, it indicates that the sample to be detected contains safrole, and the determination result is positive or weakly positive; if the detection line develops color, it indicates that the sample to be detected does not contain safrole, and the determination result is negative.

[0052] In some embodiments, in the safrole detection device, the structure of the safrole artificial antigen is shown in formula (IV), and the carrier protein is chicken egg white protein; and the safrole antibody is obtained by immunizing animals with the safrole artificial antigen with the structure shown in formula (III) and the carrier protein being bovine serum protein.

[0053] In some embodiments, in the safrole detection device, the safrole antibody is a monoclonal antibody.

[0054] The eighth aspect of the present application provides an immune analysis method for detecting safrole, which uses the safrole artificial antigen according to any one of the technical solutions and the safrole antibody according to any one of the technical solutions.

[0055] In some embodiments, in the safrole detection device, the structure of the safrole artificial antigen is shown in formula (IV), and the carrier protein is chicken egg white protein; and the safrole antibody is obtained by immunizing animals with the safrole artificial antigen with the structure shown in formula (III) and the carrier protein being bovine serum protein.

[0056] In some embodiments, the safinamide antibody is a monoclonal antibody.

[0057] In some embodiments, the immunoassay method is colloidal gold immunochromatography or ELISA.

[0058] Compared with the prior art, the present application has at least the following beneficial effects:

[0059] (1) The present application provides two kinds of safinamide haptens with suitable length of spacer arm, after coupling with carrier protein, the haptens can be fully exposed, and the recognition of spacer arm can be avoided as much as possible, so that the antibody recognizing safinamide is more optimal.

[0060] (2) The safinamide haptens are used to prepare safinamide artificial antigens and antibodies, and the safinamide monoclonal antibodies obtained have high titer, strong specificity and high affinity.

[0061] (3) The immunoassay method based on the safinamide artificial antigens and antibodies provided by the present application has good specificity for detecting safinamide, has no cross reaction with analogues of safinamide, has high sensitivity, can rapidly qualitatively and quantitatively detect safinamide in a sample, is simple to operate, and has accurate and reliable detection results.

[0062] (4) The ELISA method based on the safinamide artificial antigens and antibodies provided by the present application has a minimum detection limit LOD of 0.09 ng / mL, a half-inhibitory concentration IC 50 of 2.05 ng / mL, and a quantitative detection range of 0.28-14.89 ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0063] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments described in the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0064] Figure 1 is the ultraviolet full wavelength scanning identification diagram of BSA, SF-3 and SF-3-BSA in the embodiments of the present application;

[0065] Figure 2 is the ultraviolet full wavelength scanning identification diagram of OVA, SF-4 and SF-4-OVA in the embodiments of the present application;

[0066] Figure 3is a standard curve diagram of an ELISA method constructed by a safrole monoclonal antibody prepared by taking SF-4-OVA as a coating agent and SF-3-BSA as an immunogen in the embodiment of the present application;

[0067] Figure 4 is a structural schematic diagram of a colloidal gold test strip provided in the embodiment 5 of the present application;

[0068] Figure 5 is a test result judgment diagram of the safrole detection by the colloidal gold test strip in the embodiment 5 of the present application. DETAILED DESCRIPTION

[0069] The technical solutions of the present application are described in detail below with specific embodiments, so that the technical solutions of the present application can be better understood and implemented by the skilled in the art. The specific functional details disclosed herein should not be interpreted as limiting, but only as a basis for the claims and for teaching the skilled in the art to employ the representative basis of the present application in different ways in any appropriate detailed embodiment.

[0070] In addition, unless otherwise specified, the various raw materials used in the following embodiments can be obtained from the market or the like, and the various production and testing equipment used is known in the art, and the testing methods used are also known in the art.

[0071] Unless otherwise specified, the amount of raw materials involved in the embodiments of the present application is expressed in equivalent (eq), which refers to molar equivalent.

[0072] Embodiment 1

[0073] This embodiment provides a safrole hapten and a synthesis method thereof, and identifies the synthesized safrole hapten

[0074] 1. Synthesis of safrole hapten SF-3

[0075] The synthesis route of the safrole hapten SF-3 is as follows:

[0076]

[0077] The specific steps are as follows:

[0078] (1) Dissolve 1.2 eq of aluminum chloride and 1.2 eq of pyridine in acetonitrile, and after the heat is gone, add 1 eq of eugenol, reflux overnight; after the reaction is completed, pour the reaction liquid into an ice hydrochloric acid solution, extract with ethyl acetate and water, and then retain the organic phase; add 15 g of silica gel powder to the organic phase, and then dry it using a rotary evaporator; use the developing agent (volume ratio, petroleum ether: ethyl acetate: formic acid: 70:30:1) to purify by column chromatography to obtain 4-allyl pyrocatechol.

[0079] (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, which was then dried using a rotary evaporator; 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid methyl ester was obtained by column chromatography using a 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 an aqueous solution of 1,4-dioxane and reacted for 3 h; after the reaction was completed, the pH value was adjusted to 4 by adding hydrochloric acid; the organic phase was reserved after extraction with ethyl acetate and water, and the organic phase was dried using a rotary evaporator to obtain 5-allyl benzo[D][l,3]dioxol-2-carboxylic acid.

[0080] (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, which was then dried using a rotary evaporator; tert-butyl (5-allyl benzo[D][l,3]dioxol-2- carbonyl) aminobutyrate was obtained by column chromatography using a developing agent (volume ratio, petroleum ether: ethyl acetate: 80:25); 1 eq of tert-butyl (5-allyl benzo[D][l,3]dioxol-2-carbonyl) 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 drying using a rotary evaporator, which is the safinamide hapten SF-3.

[0081] 2. Identification of safinamide hapten SF-3

[0082] The results of the nuclear magnetic resonance hydrogen spectrum of safinamide hapten SF-3 are 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).

[0083] The mass spectrometry result of the safinin hapten SF-3 is: MS: 291, ESI-[M-H] + :290.

[0084] From the mass spectrometry and nuclear magnetic resonance results, it can be seen that the mass spectrometry result corresponds to the molecular weight of the safinin hapten, and the number of hydrogen spectrum of the nuclear magnetic resonance corresponds to the number of hydrogen spectrum on the skeleton structure of the safinin hapten, indicating that the safinin hapten is successfully prepared, which is recorded as SF-3, and the structural formula is shown as formula (I):

[0085]

[0086] 3. Synthesis of safinin hapten SF-4

[0087] The synthesis route of the safinin hapten SF-4 is as follows:

[0088]

[0089] The safinin hapten SF-4 is the precursor structure 5-allylbenzo[D][1,3]dioxolane-2-carboxylic acid obtained in the process of preparing the safinin hapten SF-3, and the preparation method is specifically described above, which will not be repeated here.

[0090] 4. Identification of safinin hapten SF-4

[0091] The nuclear magnetic resonance hydrogen spectrum result of the safinin hapten SF-4 is: 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).

[0092] The mass spectrometry result of the safinin hapten SF-4 is: MS: 206, ESI-[M-H] + :205.

[0093] From the mass spectrometry and nuclear magnetic resonance results, it can be seen that the mass spectrometry result corresponds to the molecular weight of the safinin hapten, and the number of hydrogen spectrum of the nuclear magnetic resonance corresponds to the number of hydrogen spectrum on the skeleton structure of the safinin hapten, indicating that the safinin hapten is successfully prepared, which is recorded as SF-4, and the structural formula is shown as formula (II):

[0094]

[0095] Example 2

[0096] The present example provides a synthetic antigen of safrole and a method for synthesizing the same, and identifies the synthetic synthetic antigen of safrole.

[0097] 1. Synthesis of synthetic antigen of safrole

[0098] (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 and dissolved in 100 μL DMF. The mixture was stirred at room temperature for 3 h to obtain a hapten activation solution;

[0099] (2) BSA (bovine serum albumin, 10 mg, 0.00015 mol) was dissolved in 1 mL of PBS buffer (0.01 mol / L, pH=7.4) to obtain a BSA solution. The BSA solution was stirred with the hapten activation solution at 4 °C overnight;

[0100] (3) The reaction solution was collected and transferred to a treated dialysis bag, which was then placed in 5 L of 0.01 M PBS buffer. The dialysis was carried out at 4 °C for 3 days, and the pre-cooled 0.01 M PBS buffer was replaced every 8 h;

[0101] (4) After dialysis purification, the synthetic antigen of safrole was obtained and denoted as SF-3-BSA. After concentration determination and ultraviolet spectrum scanning identification, it was stored in a -20 °C refrigerator.

[0102] According to the above basically same method, the only difference is that OVA (chicken egg white protein) is used instead of BSA, and SF-4 is used instead of SF-3 to prepare the synthetic antigen of safrole, which is denoted as SF-4-OVA.

[0103] 2. Identification of synthetic antigen of safrole

[0104] BSA, OVA, SF-3, SF-4, SF-3-BSA and SF-4-OVA were taken and identified by ultraviolet full wavelength method (200-350 nm) respectively.

[0105] Figure 1 is the ultraviolet full wavelength scanning identification chart of BSA, SF-3 and SF-3-BSA, as shown in Figure 1As shown, by comparing the maximum absorbance of each substance before and after coupling, it is found that the absorption curve of SF-3-BSA is obviously different from that of the carrier protein BSA, the absorption peak of SF-3 above 343 nm is stronger, while after coupling with BSA, the absorption peak of SF-3-BSA at 230 nm is similar to that of BSA, but at 280 nm, it is obviously higher than that of BSA, and the curve is significantly shifted compared with the relative hapten SF-3. Since all the unreacted components have been removed by dialysis after coupling, the characteristic peak of the coupling product is contributed by the drug molecules combined with the protein, indicating that the reaction product is a complex of the carrier protein BSA and SF-3.

[0106] Figure 2 are the ultraviolet full wavelength scanning identification diagrams of OVA, SF-4, SF-4-OVA, as shown in the figure, Figure 2 As shown, similarly, the absorption curve of SF-4-OVA appears a characteristic peak different from OVA and SF-4, indicating that the reaction product is a complex of the carrier protein OVA and SF-4.

[0107] 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 as shown in formula (III) and (IV):

[0108] ;

[0109] Among them, Protein is the carrier protein BSA or OVA.

[0110] Example 3

[0111] The present embodiment provides a safrole monoclonal antibody and a preparation method thereof.

[0112] 1. Animal immunization

[0113] SF-3-BSA prepared in Example 2 is used as an immunogen, 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 points on the abdomen, the vaccination dose of the antigen is 100 μg per mouse, and 0.1 ml per BALB / c female mouse; the second immunization is performed after 14 days, the immunogen is emulsified with an equal volume of Freund's incomplete adjuvant, the immunization dose is the same as that of the first immunization, and the number of booster immunization 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 of the antigen is 100 μg per mouse.

[0114] 2. Cell fusion

[0115] After sprinting for three days, the cell fusion was carried out according to the conventional PEG (polyethylene glycol, molecular weight 1450) method, and the specific steps were as follows:

[0116] a. After the mice were sacrificed by eyeball bleeding, they were immediately disinfected in 75% alcohol for about 5 min, and the spleen was taken out by sterile operation. The spleen cell suspension was obtained by gently grinding with the rubber head of a syringe and passing through a 200 mesh cell sieve, collected, centrifuged (1000 rpm, 7 min), and the spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and reserved;

[0117] b. Collect SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI1640 medium in a 5% CO2 incubator. The number of SP2 / 0 tumor cells was required to reach (1-4) × 10 7 Before fusion, the SP2 / 0 tumor cells were in the logarithmic growth phase, and the tumor cells were collected and suspended in RPMI-1640 basic culture medium for cell counting.

[0118] c. Mix the two cells and make up to 15 mL, 1000 rpm / min for 7 min, discard the supernatant. Take out the centrifuge tube, shake at 60° medium speed, and then start preparing for fusion. The fusion time is controlled as follows:

[0119] First minute: slowly add PEG;

[0120] Second minute: continue to rotate for 1 min;

[0121] Third minute: add 1 mL of basic medium;

[0122] Fourth minute: add 3 mL of basic medium;

[0123] Fifth minute: add 8 mL of basic medium;

[0124] Sixth minute: add 8 mL of basic medium;

[0125] After fusion, the cell liquid was centrifuged (1000 rpm / min, 7 min), and the incubated 200 mL HAT medium was taken out and placed in a clean bench for standby.

[0126] d. Discard the supernatant of the centrifuged hybridoma cells, and suck 2 mL of HAT medium into the centrifuge tube, gently blow it evenly, and then transfer the cell liquid in the centrifuge tube to 200 mL of HAT medium. Gently shake the medium to evenly disperse the cells, and then plate.

[0127] 3. Cell screening and cell line establishment

[0128] On the third day of cell fusion, the fusion cells were subjected to semi-replacement of RPMI-1640 screening medium, and on the fifth day, full replacement was performed with RPMI-1640 transition medium containing 20% fetal bovine serum and 2% 50x HT, and on the seventh day, the cell supernatant was subjected to screening;

[0129] The screening was divided into two steps: first, positive cell wells were screened by ic-ELISA method, and second, ic-ELISA method was used to determine the inhibition effect of the positive cells using safrole as a standard; cell wells with good inhibition to the safrole standard were selected, and subcloning was performed by limiting dilution method, and after seven days, the same method was used for detection; three times of subcloning were performed according to the above method, and finally, the safrole monoclonal antibody cell strain SF-Q2C was obtained.

[0130] 4. Preparation and identification of safrole monoclonal antibody

[0131] 8-10 week-old BALB / c mice were selected, and each mouse was injected with 1 mL of sterile paraffin oil intraperitoneally; after 7 days, each mouse was injected with 1x10 6 Safrole hybridoma cells were collected from the ascites from the seventh day, and the ascites was subjected to antibody purification by immunoaffinity chromatography; under neutral conditions, Protein G combined with IgG immunoglobulin in the ascites to make other impurities flow out and be discarded. Then, 0.01 M glycine buffer was used to desorb IgG from Protein G under acidic conditions, and high-purity IgG immunoglobulin was harvested. After adjusting the pH value of the IgG immunoglobulin solution to neutral using 0.01 M Tris-HCL buffer, dialysis and desalting were performed, and finally, high-purity safrole monoclonal antibody was obtained. The purified monoclonal antibody was stored at -20 ℃.

[0132] 5. Sensitivity evaluation of safrole monoclonal antibody (indirect competitive ELISA method)

[0133] SF-4-OVA prepared in Example 2 was used as a coating agent, diluted with a coating solution (0.05 M carbonate buffer solution, pH 9.6) to a concentration of 500 ng / mL, and 100 μL / well was used to coat a 96-well enzyme-labeled plate at 4 ℃ for overnight (12 h);

[0134] The coating solution was discarded, and PBST (0.01 M PBS, 0.06% v / v Tween-20) was used for washing twice, and then the plate was tapped dry;

[0135] 120 μL of blocking solution (PBST containing 7% skimmed milk powder) was added to each well, and blocking was performed at 37 ℃ for 1 h;

[0136] Discard the closed liquid, and tap the plate. After drying at 37℃, take out and pack with a self-sealing bag for standby use;

[0137] The above-said monoclone antibody of safrole prepared with SF-3-BSA as immunogen was diluted with PBST at a volume ratio of 1:128000; the standard sample of safrole was diluted with PBST to different concentrations, so as to obtain the standard sample dilutions of safrole 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;

[0138] 50 μL / well of the standard sample dilutions of safrole with different concentrations (three groups in parallel) were added, and then 50 μL / well of the diluted monoclone antibody of safrole was added. After incubation at 37℃ for 40 min, the plate was washed five times and tapped dry;

[0139] 100 μL / well of goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST) was added, and incubation was carried out at 37℃ for 30 min. After washing five times, the plate was tapped dry;

[0140] 100 μL / well of color developing solution was added, and color development was carried out for 10 min;

[0141] 50 μL of 10% H2SO4 solution was added to terminate the reaction, and the OD value was read at 450 nm;

[0142] ELISA standard curve drawing: B / B0 was taken as the vertical coordinate (B is the absorbance OD 450 of the standard sample of safrole with different concentrations, and B0 is the absorbance OD 450 of the blank control well), and the logarithm of the concentration of the standard sample of safrole was taken as the horizontal coordinate. The standard curve was prepared by curve fitting with the Logistic function, so as to obtain the formula of the standard curve.

[0143] Figure 3 SF-4-OVA was taken as the coating agent, and the standard curve of the ELISA method constructed with the above-said monoclone antibody of safrole was drawn, as shown in Figure 3 The minimum detection limit LOD was 0.09 ng / mL, the half-inhibitory concentration IC 50 was 2.05 ng / mL, and the quantitative detection range was 0.28-14.89 ng / mL.

[0144] 6. Specificity evaluation of the monoclone antibody of safrole

[0145] Dihydrosafrole, piperonyl ring and sesamol are safrole analogues, and the specificity of the safrole monoclonal antibody prepared in this embodiment is evaluated through cross-reaction experiments.

[0146] According to the above sensitivity evaluation method, the difference is that the safrole standard is replaced by the standard of dihydrosafrole, piperonyl ring and sesamol, and detection is carried out at the same dilution ratio, and the IC50 value of each structural analogue is obtained.

[0147] 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.

[0148] Table 1 Cross-reaction results of safrole monoclonal antibody with safrole and its analogues

[0149] ;

[0150] Note: NR represents no reaction, and no reaction means that the antibody does not recognize the analogue.

[0151] As shown in Table 1, the cross-reaction rate of the safrole monoclonal antibody to safrole is 100%, the IC50 value is 2.05 ng / mL, and there is no cross-reaction to dihydrosafrole, piperonyl ring and sesamol. It shows that the recognition ability of the antibody for detecting safrole is high, the specificity is strong, the interference of methylenedioxybenzene analogues (such as dihydrosafrole, piperonyl ring and sesamol) can be effectively excluded, and safrole can be specifically detected. 50

[0152] The above results show that the safrole monoclonal antibody prepared in the present application has excellent detection performance for safrole, high sensitivity and strong specificity.

[0153] Example 4

[0154] This embodiment provides a combination of an immunogen and a coating agent of safrole.

[0155] The serum titer and inhibition rate obtained by the indirect competitive ELISA method are used to evaluate the combination effect of the immunogen and the coating agent, using the safrole artificial antigen SF-4-OVA prepared in Example 2 as the coating agent, and the safrole monoclonal antibody prepared by using SF-3-BSA as the immunogen in Example 3. The specific operation steps are as follows:

[0156] ​1. Dilute the coating antigen with coating solution (0.05 M carbonate buffer solution, pH 9.6) to a concentration of 1000 ng / mL, coat a 96-well enzyme-labeled plate at an addition amount of 100 μL / well, and incubate in a refrigerator at 4 ℃ overnight. Discard the coating solution and wash twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v)).

[0157] 2. Add 120 μL blocking solution (PBST containing 7% skim milk) to each well, block at 37 ℃ for 1 h, discard the blocking solution, tap the plate, and dry in a drying oven at 37 ℃ for standby use.

[0158] 3. Dilute the safinin monoclonal antibody with PBST at a volume ratio of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. Meanwhile, set a blank control well (replace with PBST). Dilute 1 mg / mL safinin standard product 1000 times with PBST to obtain a safinin standard product diluent with a concentration of 1 μg / mL.

[0159] 4. Set the titer column: first add 50 μL PBST to each well, then add 50 μL safinin monoclonal antibody of different dilution ratios to each well, and finally add 50 μL PBST instead of the antibody to the last well.

[0160] 5. Set the inhibition column: first add 50 μL safinin standard product diluent to each well, then add 50 μL safinin monoclonal antibody of different dilution ratios to each well, and finally add 50 μL PBST instead of the antibody to the last well.

[0161] 6. Incubate at 37 ℃ for 40 min, and wash 5 times by tapping the plate.

[0162] 7. Add goat anti-mouse secondary antibody-HRP (diluted 5000 times with PBST), incubate at 37 ℃ for 30 min, wash 5 times by tapping the plate.

[0163] 8. Add color developing solution, and incubate for color development at 37 ℃ for 10 min.

[0164] 9. Add 10% v / v H2SO4 to terminate the reaction, and read the OD value at 450 nm. Calculate the titer and inhibition rate. The titer is the antibody dilution ratio corresponding to an OD value of about 1.0, and the inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%. 450

[0165] The combination results of the safinin immunogen and the coating antigen are shown in Table 2.

[0166] ​The 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 antigens are evaluated by using the same method as described above, and the results are shown in Table 2.

[0167] Table 2 Combination effects of different immunogens and coating antigens

[0168] ;

[0169] As shown in Table 2, the monoclonal antibody prepared by using the artificial antigen SF-3-BSA of safrole as an immunogen is the best combination of immunogens and coating antigens with SF-4-OVA, and in this combination, the monoclonal antibody can not only specifically recognize the target analyte safrole, but also has excellent antibody sensitivity.

[0170] Example 5

[0171] This example provides a colloidal gold test strip assembly for detecting safrole.

[0172] 1. Assembly of colloidal gold rapid detection test strip

[0173] Figure 4 is a structural diagram of the colloidal gold test strip of this example, as shown in Figure 4 The colloidal gold rapid detection test strip includes an NC membrane (nitrocellulose membrane), a sample pad, a water absorption pad, and a PVC plastic base plate.

[0174] The coating antigen (SF-4-OVA) is sprayed on the NC membrane by using an XYZ three-dimensional spray point film drawing instrument at a spraying amount of 0.8 μL / cm as a test line (T line); the goat anti-mouse IgG is sprayed on the NC membrane by using the same method and dose as a control line (C line); the test line (T line) and the control line (C line) are located in the middle of the NC membrane and are spaced apart from each other by 6 mm; after being dried at 37℃ for 12 h, the cellulose membrane is pasted on the middle part of the base plate, the sample pad overlaps the T line end of the NC membrane by 1 mm, and the water absorption pad is pasted on the upper side of the cellulose membrane and overlaps the cellulose membrane by 1 mm; the assembled test paper plate is cut into a test strip with a width of 3.5 mm by using a cutting machine.

[0175] 2. Preparation of gold-labeled antibody

[0176] The colloidal gold suspension with an average diameter of 30 nm is prepared by using the method of reducing chloroauric acid with trisodium citrate, and the gold-labeled antibody is prepared by using the colloidal gold to label the monoclonal antibody of safrole prepared in Example 3, and the specific method is as follows:

[0177] Take 1 mL colloidal gold solution, add 0.2 mol / L K2CO3 solution to adjust pH to about 8.0, add 10 μg of the prepared monoclonal antibody of safinin 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.

[0178] 3. Preparation of sample liquid for detection

[0179] Mix the sample to be detected with PBST at a volume ratio of 1:900 to obtain a sample liquid.

[0180] 4. Detection step

[0181] Take 120 μL of the sample liquid 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 liquid and react for 3 min, then remove the test strip and the sample pad, and determine the result after 3-5 min.

[0182] 5. Determination of detection result

[0183] Figure 5 is a test result determination chart for safinin detection using the above colloidal gold test strip, as shown in Figure 5 , if the sample does not contain the test substance safinin, the gold-labeled antibody binds to the coated antigen on the T line (test line) of the rapid test strip, making the test line show a clear red line, indicating that the detection sample is negative (such as Figure 5 in A); if the sample contains the test substance safinin, the safinin binds to the gold-labeled antibody and cannot be captured by the test line of the rapid test strip, so the test line does not develop color, indicating that it is positive (such as Figure 5 in B); similarly, the gold-labeled antibody also binds to the sheep anti-mouse IgG on the C line (control line) on the cellulose membrane, making the control line show red color, and the control line has color, indicating that the detection result of this test strip is valid (such as Figure 5 in A and B), and the control line has no color, indicating that the detection result of this test strip is invalid (such as Figure 5 in C and D).

[0184] In summary, the application provides two kinds of safinin haptens with suitable length of spacer arm, which can expose the haptens sufficiently after coupling with carrier protein, and can avoid recognition of the spacer arm as much as possible, and can produce better antibody recognizing safinin. The application also uses safinin haptens to prepare artificial antigens and antibodies, and the safinin monoclonal antibodies prepared have high titer, strong specificity and high affinity. The immunoassay constructed using the safinin artificial antigens and antibodies provided by the application has high specificity and sensitivity, and can realize rapid qualitative and quantitative detection of safinin in samples. The application simplifies the synthesis steps of the haptens, reduces the detection limit of the antibodies, and improves the specificity and sensitivity of the safinin antibodies.

[0185] Aspects, embodiments, features, and examples of the present application should be considered in all respects, as illustrative and not restrictive, the scope of the application being defined solely by the claims. Other embodiments, modifications, and uses thereof will occur to those skilled in the art upon considering the specification and may be made without departing from the spirit and scope of the application as claimed.

[0186] While the application has been described with reference to illustrative embodiments, those skilled in the art will appreciate that various other changes, omissions, and / or additions can be made thereto without departing from the spirit and scope of the application. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the application without departing from the scope thereof. Therefore, it is intended that the application not be limited to the disclosed embodiments, but that the application will include 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 denote any ordinal, but rather indicates the nature of the elements being discussed.

Claims

1. A safinin hapten, characterized in that: It has a structure as shown in formula (I) or formula (II): 。 2. The method of claim 1 for the preparation of a sahnin hapten, characterized in that, The method comprises the following steps: A first mixed reaction system containing eugenol, pyridine and aluminum halide is subjected to a first reaction at 100-120 DEG C to obtain 4-allyl pyrocatechol, which has a structure as shown in formula (I) or formula (II): ； A second mixed reaction system containing the 4-allyl pyrocatechol, methyl dichloroacetate and an acid binding agent is subjected to a second reaction at 80-100 DEG C to obtain 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid methyl ester, which has a structure as shown in formula (II): ； The 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid methyl ester is hydrolyzed under alkaline conditions, and then the pH value of the hydrolyzed reaction product is adjusted to be acidic to obtain 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid, i.e. the safrole hapten as shown in formula (II).

3. The method of claim 2, wherein: The molar equivalent of eugenol, pyridine and aluminum halide in the first mixed reaction system satisfies 1:1-3:1-2.

4. The method of claim 2, wherein: The first reaction is performed for 10-12 hours.

5. The method of claim 2, wherein: The molar equivalent of 4-allyl pyrocatechol, methyl dichloroacetate and the acid binding agent in the second mixed reaction system satisfies 1:1-3:1-3.

6. The method of claim 2, wherein: The second reaction is performed for 6-8 hours.

7. The method of claim 2, wherein: The 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid methyl ester is mixed with the base at a molar equivalent of 1:1-3 and subjected to the hydrolysis at 0-40 DEG C.

8. The method of claim 2, wherein: The pH value of the hydrolyzed reaction product is adjusted to be 2-4.

9. The preparation method according to claim 2, characterized in that, The method comprises the following steps: The 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid is mixed with N, N'-carbonyldiimidazole, and then 4-aminobutyric acid tert-butyl ester is added and subjected to a fourth reaction at 0-40 DEG C to obtain (5-allyl benzo [D] [1, 3] dioxolane-2-carbonyl) aminobutyric acid tert-butyl ester, which has a structure as shown in formula (II): ； The (5-allyl benzo [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 DEG C to obtain (5-allyl benzo [D] [1, 3] dioxolane-2-carbonyl) aminobutyric acid, i.e. the safrole hapten as shown in formula (I).

10. The method of claim 9, wherein: The molar equivalent of the 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid, N, N'-carbonyldiimidazole and 4-aminobutyric acid tert-butyl ester satisfies 1:1-2:1-2.

11. The method of claim 9, wherein: The 5-allyl benzo [D] [1, 3] dioxolane-2-carboxylic acid is first mixed with N, N'-carbonyldiimidazole for 0.5-2 hours, and then the 4-aminobutyric acid tert-butyl ester is added and reacted for 2-4 hours.

12. The method of claim 9, wherein: The molar equivalent of the (5-allyl benzo [D] [1, 3] dioxolane-2-carbonyl) aminobutyric acid tert-butyl ester and the acid satisfies 1:1-3.

13. The method of claim 9, wherein: The fifth reaction is performed for 4-6 hours.

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

15. The artificial antigen of safflower according to claim 14, characterized in that: The carrier protein is any one of bovine serum albumin, chicken egg albumin, hemocyanin and lactoferrin.

16. A combination of artificial antigens of safinin, characterized in that, The immunogen is the artificial antigen of safflower yellow A as claimed in claim 14, and its structure is shown as formula (III) and the carrier protein is bovine serum albumin; and the coating antigen is the artificial antigen of safflower yellow A as claimed in claim 14, and its structure is shown as formula (IV) and the carrier protein is chicken egg albumin.

17. The use of the safflower yellow A hapten as claimed in claim 1, the artificial antigen of safflower yellow A as claimed in claim 14 or 15, or the artificial antigen of safflower yellow A as claimed in claim 16 in the preparation of a safflower yellow A detection product or in the detection of safflower yellow A for the purpose of non-disease treatment diagnosis.

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