A hapten, an artificial antigen, and their applications for detecting baicalin, baicalein, wogonin, and baicalein.

Abstract

This invention provides a hapten, an artificial antigen, and their applications for detecting baicalin, baicalein, wogonin, and baicalein. The detection method constructed using the artificial antigen of this invention exhibits high detection sensitivity for baicalin, with an IC50 value of [missing value]. 50 The concentration was 13.35 ng / mL, the limit of detection (LOD) was 0.38 ng / mL, and the linear range was 1.42–125.52 ng / mL. This method also exhibits broad-spectrum recognition of baicalin, wogonin, and scutellarin in Scutellaria baicalensis, with an IC50 value of 13.35 ng / mL. 50 All values ​​were less than 1 μg / mL. This invention effectively overcomes the limitations of existing technologies that rely on a single target for evaluating the detection of illegal additives in Scutellaria baicalensis, significantly improving the comprehensiveness of the detection and evaluation, and has broad application prospects in the field of screening and quality control of illegal additives in health foods.

Description

Technical Field

[0001] This invention relates to the technical field of safety testing for health products, specifically to a hapten, an artificial antigen, and their applications for detecting baicalin, baicalein, wogonin, and wogonin. Background Technology

[0002] Scutellaria baicalensis, a commonly used traditional Chinese medicine, primarily contains flavonoids, mainly baicalin, baicalein, wogonin, and baicalein glycosides. Baicalin is its core active substance, possessing anti-inflammatory and antioxidant pharmacological effects. However, due to the complex composition of Scutellaria baicalensis, there are significant blind spots in the detection of its related components, difficulties in accurately identifying detection targets, and existing detection methods often being limited to single targets. This leads to oversights in the regulatory process, providing opportunities for unscrupulous individuals to illegally add Scutellaria baicalensis-related components not listed in the "List of Substances That are Traditionally Both Food and Chinese Medicinal Herbs" to health products. This behavior poses a serious hidden threat to the health of consumers.

[0003] According to the Pharmacopoeia of the People's Republic of China, Scutellaria baicalensis and baicalin are clearly classified as drugs. The Food Safety Law of the People's Republic of China requires that no drug ingredients be added to food. Scutellaria baicalensis and its related active ingredients (including baicalin, baicalein, wogonin, etc.) have never been approved by relevant national departments for use as food-medicine homologous substances, food additives, new food raw materials, or health food raw materials in the production of health products. Therefore, ordinary food and health products should not contain any components of Scutellaria baicalensis. In addition, the group standard "Determination of the content of seven flavonoids in Scutellaria baicalensis by high performance liquid chromatography" (T / NAIA 0348-2024) has made clear requirements for the detection limits (LOD of 0.075 mg / kg) of the main active substances in Scutellaria baicalensis (baicalin, wogonin, baicalein, and wogonin), providing a certain reference for the detection of related components in Scutellaria baicalensis, but it still cannot quickly solve the need for comprehensive detection of multiple components in Scutellaria baicalensis.

[0004] Scutellaria baicalensis itself poses significant safety risks. Clinical studies and research have shown that it can cause rare adverse reactions such as liver damage, jaundice, and interstitial pneumonia. It can also directly affect the gut microbiota, and long-term, high-dose use can easily lead to gut microbiota dysbiosis, resulting in intestinal mucosal damage and decreased intestinal immune function. Even small doses can cause sensitization and toxic reactions such as bullous drug eruptions and a sudden drop in white blood cells. Given these safety risks, the immunotoxicity and overall safety of Scutellaria baicalensis cannot be ignored. Using baicalin as a single target for quality control is insufficient to comprehensively and accurately assess the safety risks of related components of Scutellaria baicalensis, and it is also difficult to meet regulatory requirements.

[0005] Currently, for the detection of Scutellaria baicalensis, relevant technologies have been developed, including baicalin haptens, antibodies, and corresponding immunoassay methods. However, existing rapid detection technologies all use antibodies prepared by directly conjugating baicalin with carrier proteins, which suffers from low potency and limited detection targets. This makes it impossible to comprehensively detect multiple active components (baicalin, baicalein, wogonin, and baicalein) in Scutellaria baicalensis, failing to meet the comprehensive regulatory needs for the illegal addition of Scutellaria baicalensis in the health supplement industry. In on-site testing scenarios, highly potent antibodies can effectively reduce the dosage used per test, lower production costs, and ensure the reliability of test results. There are currently no reports on the use of baicalin antibodies for the broad-spectrum rapid detection of multiple targets (baicalin, baicalein, wogonin, and baicalein) in Scutellaria baicalensis in the health supplement industry.

[0006] Therefore, it is particularly urgent and necessary to develop a hapten and artificial antigen that can accurately identify the above-mentioned active ingredients of Scutellaria baicalensis, and then establish an efficient and comprehensive immunoassay method for the illegal addition of Scutellaria baicalensis to health products, so as to solve the shortcomings of existing detection technologies and meet the needs of supervision and food safety assurance. Summary of the Invention

[0007] To overcome the aforementioned defects and deficiencies in the existing technology, the present invention provides a hapten, an artificial antigen, and their applications for detecting baicalin, baicalein, wogonin, and wogonin.

[0008] The first objective of this invention is to provide a hapten of baicalin, baicalein, wogonin and / or wogonin.

[0009] A second object of the present invention is to provide the use of the above-mentioned hapten in the preparation of artificial antigens of baicalin, baicalein, wogonin and / or wogonin.

[0010] A third objective of this invention is to provide an artificial antigen of baicalin, baicalein, wogonin and / or wogonin.

[0011] A fourth object of the present invention is to provide the use of the above-mentioned artificial antigen in the preparation of antibodies against baicalin, baicalein, wogonin and / or wogonin.

[0012] The fifth objective of this invention is to provide an artificial antigen combination.

[0013] A sixth object of the present invention is to provide the use of the above-described artificial antigen combination in the preparation of products for detecting baicalin, baicalein, wogonin and / or wogonin.

[0014] A seventh object of the present invention is to provide a kit for detecting baicalin, baicalein, wogonin and / or wogonin.

[0015] The eighth object of the present invention is to provide a method for detecting baicalin, baicalein, wogonin and / or wogonin.

[0016] This invention claims protection for the following: A hapten of baicalin, baicalein, wogonin and / or wogonin, the structural formula of said hapten being shown in formula (V).

[0017] Formula (V); Where n is 1 to 4.

[0018] Preferably, n=3, and the structural formula of the hapten is shown in formula (Ⅰ).

[0019] Formula (I); Or, if n=1, the structural formula of the hapten is as shown in formula (II).

[0020] Formula (II).

[0021] The above-mentioned hapten is used in the preparation of artificial antigens of baicalin, baicalein, wogonin and / or wogonin.

[0022] An artificial antigen of baicalin, baicalein, wogonin and / or wogonin, said artificial antigen being obtained by conjugating the above-mentioned hapten with a carrier protein, the structural formula of which is shown in formula (VI).

[0023] Formula (VI); Wherein, P is the carrier protein.

[0024] Preferably, the carrier protein is bovine serum albumin, n is 3, and its structural formula is shown in formula (Ⅲ).

[0025] Formula (Ⅲ); Alternatively, the carrier protein may be chicken ovalbumin, where n is 1, and its structural formula is shown in formula (Ⅳ).

[0026] Formula (Ⅳ).

[0027] The above-mentioned artificial antigens are used in the preparation of antibodies against baicalin, baicalein, wogonin and / or wogonin.

[0028] An antibody against baicalin, baicalein, wogonin and / or wogonin is prepared by immunizing animals with an artificial antigen obtained by conjugating the hapten shown in formula (I) with bovine serum albumin.

[0029] An artificial antigen combination comprising an immunogen and a coating antigen; The immunogenicity is obtained by conjugating the hapten shown in formula (Ⅰ) with bovine serum albumin, where n=3; The coating is obtained by conjugating chicken oocyte albumin with the hapten shown in formula (II), where n=1.

[0030] The above-mentioned artificial antigen combination is used in the preparation of products for detecting baicalin, baicalein, wogonin and / or wogonin.

[0031] A kit for detecting baicalin, baicalein, wogonin and / or wogonin, the kit comprising the above-described artificial antigen combination.

[0032] A method for detecting baicalin, baicalein, wogonin and / or wogonin, using the above-mentioned artificial antigen combination for detection, said detection being for the purpose of non-disease treatment diagnosis.

[0033] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a hapten, an artificial antigen, and their applications for detecting baicalin, baicalein, wogonin, and baicalein. The detection method constructed using the artificial antigen of this invention exhibits high detection sensitivity for baicalin, with an IC50 value of [missing value]. 50 The concentration was 13.35 ng / mL, the limit of detection (LOD) was 0.38 ng / mL, and the linear range was 1.42–125.52 ng / mL. This method also exhibits broad-spectrum recognition of baicalin, wogonin, and scutellarin in Scutellaria baicalensis, with an IC50 value of 13.35 ng / mL. 50 The values ​​were 22.91 ng / mL, 51.72 ng / mL, and 66.16 ng / mL, respectively; the limits of detection (LODs) were 1.33 ng / mL, 3.18 ng / mL, and 5.51 ng / mL, respectively; and the linear ranges were 3.81–137.77 ng / mL, 8.90–300.52 ng / mL, and 13.79–317.32 ng / mL, respectively. This invention effectively overcomes the limitations of existing technologies that use only a single target for evaluating the detection of illegal additives in Scutellaria baicalensis, significantly improving the comprehensiveness of the detection and evaluation, and has broad application prospects in the field of screening and quality control of illegal additives in health foods. Attached Figure Description

[0034] Figure 1 This is a synthetic route diagram for the hapten H-4C.

[0035] Figure 2 This is a synthetic route diagram for the hapten H-2C.

[0036] Figure 3 The image shows the results of ultraviolet full-wavelength scanning identification of carrier protein BSA, hapten H-4C, and artificial antigen H-4C-BSA.

[0037] Figure 4 The image shows the results of ultraviolet full-wavelength scanning identification of carrier protein OVA, hapten H-2C, and artificial antigen H-2C-OVA.

[0038] Figure 5 This is a standard curve for an indirect competitive ELISA using antibodies to detect baicalin. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Example 1: Synthesis and Identification of Hapten I. Experimental Methods 1. Synthesis and identification of hapten H-4C The synthetic route diagram of the hapten H-4C is as follows: Figure 1 As shown, the specific steps include: Weigh 200 mg of 7-hydroxyflavone (1 equivalent) and 300 mg of tert-butyl 4-bromobutyrate (1.5 equivalent) into a 50 mL round-bottom flask, dissolve in 10 mL of DMF, add 230 mg of potassium carbonate (2 equivalent) and 10 mg of sodium iodide, stir at room temperature until the solid dissolves, and react at 50 °C overnight.

[0042] The next day, after the reaction cooled to room temperature, the crude product was extracted with a solution of water and ethyl acetate in a 1:4 volume ratio to remove potassium carbonate and sodium iodide. The organic phase was collected and concentrated. 2 mL of dichloromethane and 2 mL of trifluoroacetic acid were added to the concentrated organic phase for hydrolysis. The mixture was stirred at room temperature for 60 min. After the reaction was monitored using a TCL plate, NaOH solution was added to remove excess trifluoroacetic acid. A solution of water and ethyl acetate in a 1:4 volume ratio was added for back-extraction, and the aqueous phase was collected. HCl solution was added to the collected aqueous phase to adjust the pH to 5.0. The mixture was then extracted again with a solution of water and ethyl acetate in a 1:4 volume ratio, and the organic phase was collected and concentrated. 5 mL of developing solvent (V...) was added... 二氯甲烷 V 甲醇 V甲酸 The organic phase was dissolved and concentrated using a ratio of 20:1:1, and then purified by column chromatography to obtain the hapten H-4C.

[0043] The hapten H-4C was identified by proton nuclear magnetic resonance spectroscopy and mass spectrometry.

[0044] 2. Synthesis and identification of hapten H-2C The synthetic route diagram of the hapten H-2C is as follows: Figure 2 As shown, the specific steps include: Weigh 200 mg of 7-hydroxyflavone (1 equivalent) and 350 mg of tert-butyl 2-bromoacetate (1.5 equivalent) into a 50 mL round-bottom flask, dissolve in 10 mL of DMF, add 230 mg of potassium carbonate (2 equivalent) and 10 mg of sodium iodide, stir at room temperature until the solid dissolves, and react at 50 °C overnight.

[0045] The next day, after the reaction cooled to room temperature, the crude product was extracted with a solution of water and ethyl acetate in a 1:4 volume ratio to remove potassium carbonate and sodium iodide. The organic phase was collected and concentrated. 2 mL of dichloromethane was added to the concentrated organic phase for dissolution, and 2 mL of trifluoroacetic acid was added for hydrolysis. The reaction was stirred at room temperature for 60 min. After the reaction was monitored using a TCL plate, NaOH solution was added to remove excess trifluoroacetic acid. A solution of water and ethyl acetate in a 1:4 volume ratio was added for back-extraction, and the aqueous phase was collected. HCl solution was added to the collected aqueous phase to adjust the pH to 5.0. The mixture was then extracted again with a solution of water and ethyl acetate in a 1:4 volume ratio, and the organic phase was collected and concentrated. 5 mL of developing solvent (V...) was added... 二氯甲烷 V 甲醇 V 甲酸 The organic phase was dissolved and concentrated using a ratio of 20:1:1, and then purified by column chromatography to obtain the hapten H-2C.

[0046] The hapten H-2C was identified by proton nuclear magnetic resonance spectroscopy and mass spectrometry.

[0047] II. Experimental Results The results of the 1H NMR spectrum identification of the hapten H-4C are as follows: 1 H NMR (600 MHz, Methanol- d 4) δ8.08 – 8.02 (m, 2H), 7.71 (d, J = 9.1 Hz, 1H), 7.62 – 7.53 (m, 4H), 7.44 (dd, J =9.1, 3.1 Hz, 1H), 6.92 (s, 1H), 4.15 (t, J= 6.2 Hz, 2H), 2.53 (t, J = 7.3 Hz, 2H), 2.17 – 2.11 (m, 2H). The mass spectrometry results for the hapten H-4C are as follows: MS: C 19 H 16 O5: 324.10, ESI-[MH] - :323.10.

[0048] Based on the results of the proton nuclear magnetic resonance spectrum and mass spectrometry, it can be determined that the hapten H-4C has been successfully synthesized, and its structural formula is shown in formula (Ⅰ):

[0049] Formula (I).

[0050] The hapten H-4C was named 4-((4-oxo-2-phenyl-4H-benzopyran-7-yl)oxy)butyric acid using systematic nomenclature.

[0051] The results of the 1H NMR spectrum identification of the hapten H-2C are as follows: 1 H NMR (600 MHz, Methanol- d 4) δ8.06 (dd, J = 8.1, 1.6 Hz, 2H), 7.75 (d, J = 9.1 Hz, 1H), 7.64 – 7.55 (m, 3H), 7.52 (s, 1H), 6.93 (s, 2H), 4.82 (s, 2H). The mass spectrometry results for the hapten H-2C are as follows: MS: C 17 H 12 O5: 296.07, ESI-[MH] - 295.07.

[0052] The results of nuclear magnetic resonance hydrogen spectrum and mass spectrometry show that the hapten H-2C has been successfully synthesized, and its structural formula is shown in formula (II):

[0053] Equation (II).

[0054] The hapten H-2C was named 2-((4-oxo-2-phenyl-4H-benzopyran-7-yl)oxy)acetic acid using systematic nomenclature.

[0055] Example 2: Synthesis and Identification of Artificial Antigens I. Experimental Methods The hapten H-4C (structural formula shown in formula (I)) or hapten H-2C (structural formula shown in formula (II)) prepared in Example 1 was conjugated with bovine serum albumin (BSA) or chicken ovalbumin (OVA) via the active ester method. The specific steps are as follows: Take 1 mol of hapten H-4C (structural formula shown in formula (Ⅰ)), 1.0 mol of N-hydroxysuccinimide (NHS) and 1.5 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and dissolve them in 200 μL of N,N-dimethylformamide (DMF). Stir at room temperature (25℃) in the dark for 4 h to obtain the hapten H-4C activated solution, which is denoted as solution A. Dissolve 10 mg of BSA in 1 mL of coating buffer (0.01 mol / L, pH=7.4) to obtain a BSA solution, denoted as solution B. 200 μL of solution A was slowly added dropwise to 1 mL of solution B, and the reaction was carried out at 4°C for 12 h. The mixture was dialyzed with PBS buffer for 3 days, 3 times a day. After the dialyzing was completed, the artificial antigen H-4C-BSA was obtained, aliquoted into centrifuge tubes, and stored at -20°C for use.

[0056] The PBS buffer formula is as follows: 14.5 g Na2HPO4•12H2O, 42.5 g NaCl, 1.0 g KCl, 1.0 g KH2PO4, and distilled water is added to bring the volume to 500 mL.

[0057] The preparation of artificial antigen H-2C-OVA is the same as that of artificial antigen H-4C-BSA, except that OVA is used instead of BSA, and the hapten H-2C (structure shown in formula (II)) prepared in Example 1 is used instead of hapten H-4C (structure shown in formula (I)).

[0058] Artificial antigens H-4C-BSA and H-2C-OVA were identified by ultraviolet full-wavelength (200–350 nm) scanning.

[0059] II. Experimental Results The UV full-wavelength scan results of the carrier protein BSA, the hapten H-4C (structure shown in formula (Ⅰ)) and the artificial antigen H-4C-BSA are as follows: Figure 3As shown, by comparing the highest absorbance values ​​of each substance before and after coupling, it was found that the absorption curve of the artificial antigen H-4C-BSA was significantly different from that of the carrier protein BSA. The hapten H-4C (structure shown in formula (I)) had a characteristic peak at 260 nm and 300 nm, while after coupling with the carrier protein BSA, the absorption peaks of the artificial antigen H-4C-BSA at 260 nm and 300 nm were significantly higher than those of the hapten H-4C (structure shown in formula (I)), and the curve of the artificial antigen H-4C-BSA was significantly shifted relative to that of the carrier protein BSA. Since all unreacted drugs and other small molecules were removed by dialysis during the coupling reaction, the drug characteristic peaks of the coupling product were contributed by the protein-bound drug molecules, indicating that the coupling product was a complex of the carrier protein BSA and the hapten H-4C (structure shown in formula (I)). That is, the artificial antigen H-4C-BSA was successfully prepared, and its structure is shown in formula (III), where P is the carrier protein BSA:

[0060] Formula (III).

[0061] The UV full-wavelength scan results of the carrier protein OVA, the hapten H-2C (structural formula shown in formula (II)) and the artificial antigen H-2C-OVA are as follows: Figure 4 As shown, by comparing the highest absorbance values ​​of each substance before and after coupling, it was found that the absorption curve of the artificial antigen H-2C-OVA was significantly different from that of the carrier protein OVA. The hapten H-2C (structure shown in formula (II)) had a characteristic peak at 270 nm and 300 nm, while after coupling with the carrier protein OVA, the absorption peaks of the artificial antigen H-2C-OVA at 270 nm and 300 nm were significantly higher than those of the hapten H-2C (structure shown in formula (II)), and the curve of the artificial antigen H-2C-OVA was significantly shifted relative to that of the carrier protein OVA. Since all unreacted drugs and other small molecules were removed by dialysis during the coupling reaction, the drug characteristic peaks of the coupling product were contributed by the protein-bound drug molecules, indicating that the coupling product is a complex of the carrier protein OVA and the hapten H-2C (structure shown in formula (II)). That is, the artificial antigen H-2C-OVA was successfully prepared, and its structure is shown in formula (IV), where P is the carrier protein OVA:

[0062] Formula (Ⅳ).

[0063] Example 3 Preparation of Monoclonal Antibodies The artificial antigen H-4C-BSA (structural formula shown in formula (III), where P is bovine serum albumin) prepared in Example 2 was used as the immunogen and mixed with an equal volume of Freund's complete adjuvant. After thorough emulsification, Balb / c female mice were immunized by subcutaneous injection at multiple sites in the abdomen. A second immunization was performed 14 days later, using an equal volume of Freund's incomplete adjuvant mixed with the immunogen. The number of booster immunizations was 3. One week after each booster immunization, blood was collected from the tail to determine the antiserum titer. When the titer stabilized, mice with the best effect were selected for shock immunization. Three days later, B cells from the spleen of the mice were fused with myeloma cells. After 3-4 rounds of limiting dilution, the resulting positive hybridoma cells were injected intraperitoneally into mice. Ascites fluid from the mice was collected and purified to obtain monoclonal antibodies.

[0064] Example 4: Optimal combination selection of baicalin immunogen and coating antigen I. Experimental Methods Artificial antigen H-4C-OVA and artificial antigen H-2C-BSA were synthesized according to Example 2, wherein: The preparation of artificial antigen H-4C-OVA (structural formula as shown in formula (III), where P is chicken ovalbumin) is the same as that of artificial antigen H-4C-BSA (structural formula as shown in formula (III), where P is bovine serum albumin), except that OVA is used instead of BSA. The preparation of artificial antigen H-2C-BSA (structural formula as shown in formula (Ⅳ), where P is bovine serum albumin) is the same as that of artificial antigen H-4C-BSA (structural formula as shown in formula (Ⅲ), where P is bovine serum albumin), except that the hapten H-2C (structural formula as shown in formula (Ⅱ)) prepared in Example 1 is used instead of hapten H-4C (structural formula as shown in formula (Ⅰ)).

[0065] According to Example 3, monoclonal antibody 1 was prepared using artificial antigen H-2C-BSA as an immunogen.

[0066] Using artificial antigen H-4C-OVA (structural formula shown in formula (III), where P is chicken ovalbumin), monoclonal antibody 1, artificial antigen H-2C-OVA prepared in Example 2 (structural formula shown in formula (IV), where P is chicken ovalbumin), and monoclonal antibody prepared in Example 3, the titer and inhibition rate of the obtained antiserum were detected by ELISA, thereby selecting the optimal combination of immunogen and coating antigen. The specific operating steps are as follows: (1) Dilute the artificial antigen H-4C-OVA (structural formula as shown in formula (III), where P is chicken ovalbumin) or the artificial antigen H-2C-OVA (structural formula as shown in formula (IV), where P is chicken ovalbumin) with coating buffer (0.05 M carbonate buffer, pH 9.6) to a concentration of 1 μg / mL, coat 96-well microplates, add 100 μL to each well, incubate overnight in a constant temperature water bath at 37℃, discard the coating buffer, and wash twice with PBST (0.01 M PBS, 0.06% Tween-20, v / v); (2) Add 120 μL of blocking solution (6% skim milk powder solution, w / v) to each well, block at 37℃ for 3 h, discard the blocking solution, pat the plate, and dry in a drying oven at 37℃ for later use; (3) Dilute the monoclonal antibody 1 or the monoclonal antibody obtained in Example 3 with PBST at volume ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, and set up blank control wells (using PBST instead); dilute 1 mg / mL baicalin with PBST 1000 times to a concentration of 1 μg / mL; Titer series: First, add 50 μL of PBST to each well, then add 50 μL of serially diluted monoclonal antibody diluent to each well, and finally add 50 μL of PBST to the last well instead of the monoclonal antibody diluent. Inhibition column: First, add 50 μL of baicalin drug diluent to each well, then add 50 μL of serially diluted monoclonal antibody diluent to each well, and add 50 μL of PBST to the last well instead of the monoclonal antibody; incubate at 37°C for 40 min, wash 5 times, and plate. (4) Add goat anti-rabbit secondary antibody Ig-HRP (5000 times dilution), incubate at 37℃ for 30 min, wash 5 times, and plate. (5) Add color developing solution and incubate at 37°C for 10 min; (6) The reaction was terminated by adding 10% H2SO4 solution (v / v), and the OD value was read at 450 nm; Valence is OD 450 This refers to the antiserum dilution factor corresponding to approximately 1.0. Inhibition rate = (OD value of potency - OD value of inhibition) / OD value of inhibition × 100%.

[0067] II. Experimental Results The titers and inhibition rates of the antiserums combining the immunogen and coating antigen are shown in Table 1. Table 1 shows that the combination of artificial antigen H-4C-BSA (structural formula shown in formula (III), where P is bovine serum albumin) as the immunogen and artificial antigen H-2C-OVA (structural formula shown in formula (IV), where P is chicken ovalbumin) as the coating antigen exhibited the best titer (1:32000) and the highest inhibition rate (74.97%), making it the optimal combination of immunogen and coating antigen.

[0068] Table 1. Potency and inhibition rate of antiserum containing immunogen and coating antigen combination.

[0069] Example 5: Establishment of an indirect competitive ELISA method for the detection of baicalin, baicalein, wogonin, or wogonin in Scutellaria baicalensis. I. Experimental Methods This embodiment provides an indirect competitive ELISA method for detecting baicalin, baicalein, wogonin, or baicalein in Scutellaria baicalensis, specifically including the following steps: (1) The artificial antigen H-2C-OVA (structural formula as shown in formula (Ⅳ), where P is chicken ovalbumin) prepared in Example 2 was used as the coating antigen. It was diluted with coating solution to a concentration of 500 ng / mL and coated with 96-well microplate. 100 μL was added to each well and incubated at 37°C overnight (12 h). (2) Discard the coating solution, wash twice, and pat dry; (3) Add 120 μL of blocking solution (6% skim milk powder solution, w / v) to each well and block at 37℃ for 3 h; (4) Discard the sealing liquid, pat the plate, dry at 37°C for 30 min, and then pack it in a self-sealing bag for later use; (5) The monoclonal antibody obtained in Example 3 was diluted with PBST at volume ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000. Blank control wells were set up (replaced with PBST). 1 mg / mL of the drug (baicalin, baicalein, wogonin or wogonin) was diluted 1000 times with PBST to a concentration of 1 μg / mL. (6) Add 50 μL of drug diluent to each row, then add 50 μL of monoclonal antibody diluent per well, incubate at 37°C for 40 min, wash five times, and pat dry; (7) Add 100 μL of goat anti-rabbit secondary antibody-HRP (5000-fold dilution) to each well, incubate at 37℃ for 30 min, wash five times, and pat dry; (8) Add 100 μL of colorimetric solution to each well and develop the color for 10 min; (9) Add 50 μL of 10% H2SO4 solution (v / v) to terminate the reaction, and read the OD value at 450 nm to obtain the titer and inhibition rate. The titer is OD. 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%; (10) Construction of ELISA standard curve: The logarithm of the concentration of the drug standard is used as the abscissa, and B / B0 is used as the ordinate (B is the absorbance value OD450 of different concentrations of drug standard, and B0 is the absorbance value OD450 of the blank control well). The Logistic function is used for curve fitting to obtain the standard curve, and the formula of the standard curve and IC are obtained. 50 value.

[0070] II. Experimental Results Depend on Figure 5 It can be seen that the half-maximal inhibitory concentration (IC50) of the monoclonal antibody prepared in Example 3 against baicalin is... 50 The concentration was 13.35 ng / mL, and the limit of detection (LOD) was 13.35 ng / mL. 10 The concentration of baicalin was 0.38 ng / mL, and the linear range was 1.42–125.52 ng / mL, indicating that the monoclonal antibody prepared in this invention has high sensitivity in detecting baicalin and can meet the detection requirements.

[0071] Furthermore, Table 2 shows that the indirect competitive ELISA detection method established using the monoclonal antibody and coating antigen prepared in this invention also has broad-spectrum recognition ability for baicalin, wogonin, and wogonin in Scutellaria baicalensis, with high sensitivity and IC50. 50 All levels were below 1 μg / mL, meeting the requirements for qualitative detection applications.

[0072] Table 2. Detection results of baicalin, baicalein, wogonin, or wogonin in Scutellaria baicalensis.

[0073] Example 6: ELISA kit for detecting baicalin, baicalein, wogonin, and baicalein in Scutellaria baicalensis 1. Components of the reagent kit (1) Preparation of enzyme-labeled plates coated with coating antigen ① The artificial antigen H-2C-OVA (structural formula as shown in formula (Ⅳ), where P is chicken ovalbumin) prepared in Example 2 was used as the coating antigen. The coating antigen was diluted to a concentration of 500 ng / mL with coating buffer and coated with a 96-well microplate. Then 100 μL was added to each well and incubated overnight at 37°C in the dark. ② Pour out the liquid from the hole, wash twice with washing solution for 30 seconds each time, and pat dry; ③ Add 200 μL of blocking solution to each well, incubate at 25°C in the dark for 2 h, pour out the liquid in the well, pat dry, and then vacuum seal with aluminum foil for storage. Coating buffer: 0.05 M carbonate buffer with a pH of 9.6; Blocking solution: 6% skim milk powder solution (w / v).

[0074] (2) Standard products Ten drug standards with different concentration gradients (baicalin, baicalein, wogonin, and wogonein) were prepared, with concentrations of 62,500 ng / mL, 12,500 ng / mL, 2,500 ng / mL, 100 ng / mL, 20 ng / mL, 4 ng / mL, 0.8 ng / mL, 0.16 ng / mL, 0.032 ng / mL, and 0.0064 ng / mL, respectively.

[0075] (3) Antibody Monoclonal antibody prepared in Example 3.

[0076] (4) Enzyme conjugates Horseradish peroxidase-labeled goat anti-rabbit secondary antibody or horseradish peroxidase-labeled goat anti-mouse secondary antibody.

[0077] (5) Substrate developing solution It consists of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine.

[0078] (6) Termination solution 2 mol / L H2SO4.

[0079] (7) Washing liquid pH 7.4, 0.8% Tween-20 (v / v), 0.02% sodium azide preservative (w / v), 0.2 M phosphate buffer; Before use, dilute the detergent solution 20 times with water (1 part detergent solution to 19 parts water, prepare fresh before use) to obtain the working solution.

[0080] (8) Sealing liquid 6% defatted milk powder solution (w / v).

[0081] 2. How to use (1) Sample testing Number the corresponding wells of the samples and standards sequentially, and prepare two parallel wells for each sample and standard, recording the positions of the standard and sample wells. Dilute the enzyme conjugate concentrate with enzyme conjugate diluent at a volume ratio of 1:10 as needed (i.e., add one part enzyme conjugate concentrate to 10 parts enzyme conjugate diluent, prepare fresh before use).

[0082] Add 50 μL of standard / sample to the corresponding well, then add 50 μL of antibody working solution, gently vortex to mix, cover with a cover film, and incubate at 25°C in the dark for 40 min. Shake off the liquid in the wells, add 250 μL of washing working solution per well; wash thoroughly 4–5 times, with 10 s intervals between each wash, discard the washing solution, and pat dry with absorbent paper (any remaining air bubbles can be popped with an unused pipette tip). Add 50 μL of substrate chromogenic solution A per well, then add 50 μL of substrate chromogenic solution B per well, gently vortex to mix, cover with a cover film, and incubate at 25°C in the dark for 10 min. Add 50 μL of stop solution per well, gently vortex to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0083] (2) Analysis of test results 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 Using the logarithm of the concentration of the drug standard as the abscissa, a logistic function was used for curve fitting to obtain a standard curve, and the formula for the standard curve was derived. The OD of the sample was then... 450 Substituting the average value into the formula of the standard curve above, we obtain the concentration of the sample. Then, multiplying it by the corresponding dilution factor, we obtain the actual concentrations of baicalin, baicalein, wogonin, and baicalein in the tested sample of Scutellaria baicalensis.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A hapten of baicalin, baicalein, wogonin, and / or wogonin, characterized in that, The structural formula of the hapten is shown in formula (V). Formula (V); Where n is 1 to 4.

2. The hapten according to claim 1, characterized in that, The value of n is 1 or 3.

3. The use of the hapten according to claim 1 or 2 in the preparation of artificial antigens of baicalin, baicalein, wogonin and / or wogonin.

4. An artificial antigen of baicalin, baicalein, wogonin, and / or wogonin, characterized in that, The artificial antigen is obtained from the hapten-coupled carrier protein according to claim 1 or 2, and its structural formula is shown in formula (VI). Formula (VI); Wherein, P is the carrier protein.

5. The artificial antigen according to claim 4, characterized in that, The carrier protein is bovine serum albumin or chicken oocyte albumin.

6. The use of the artificial antigen according to any one of claims 4 or 5 in the preparation of antibodies against baicalin, baicalein, wogonin and / or wogonin.

7. A combination of artificial antigens, characterized in that, Includes immunogens and coating antigens; The immunogen is obtained by conjugating the hapten with bovine serum albumin as described in claim 1, wherein n=3; The coating is obtained by conjugating the hapten with chicken oocyte albumin as described in claim 1, wherein n=1.

8. The use of the artificial antigen combination according to claim 7 in the preparation of products for detecting baicalin, baicalein, wogonin and / or wogonin.

9. A kit for detecting baicalin, baicalein, wogonin and / or wogonin, characterized in that, The kit contains the artificial antigen combination as described in claim 7.

10. A method for detecting baicalin, baicalein, wogonin, and / or wogonin, characterized in that, The detection is performed using the artificial antigen combination as described in claim 7, wherein the detection is for the purpose of non-disease treatment diagnosis.

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