Diosmin antigen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of compound preparation and application, in particular to a diosmin antigen and a preparation method and application thereof. According to the present invention, the diosmin antigen is prepared, the diosmin antibody is obtained through the diosmin antigen, and the diosmin antigen and the antibody are used for diosmin detection in the environment, such that compared with the prior art, the diosmin antigen can be conveniently and rapidly obtained, the synthesis steps are concise, the synthesis cost is low, and the effect is good. According to the present invention, the diosmin antibody with characteristics of good specificity and high sensitivity can be obtained by immunizing through the diosmin antigen, the diosmin detection can be simply, conveniently and rapidly performed, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of compound preparation and application, and in particular to a diosmin antigen and a preparation method and application thereof. Background Art

[0002] Diosmin, also known as diosmin, has a common name of diosmin. CAS registration number: 520-27-4; molecular formula: C 28 H 32 O 15 ; Relative molecular weight: 608.54. The structural formula is as follows:

[0003]

[0004] Diosmin was first isolated from Scrophularianodosa in 1925. It can also be obtained from the natural product hesperidin by semi-synthetic methods. It is a typical flavonoid compound that is found in large quantities in citrus fruits. It is mainly used clinically to treat the following diseases: symptoms related to venous lymphatic insufficiency and symptoms of acute hemorrhoid attacks. In some other studies, diosmin also showed antioxidant, anti-inflammatory, liver protection, blood sugar regulation, and nerve pain relief effects.

[0005] At present, the extraction methods of diosmin mainly include ultrasonic extraction, Soxhlet extraction, response surface methodology optimized ultrasonic extraction, solid phase dispersion extraction, high-speed countercurrent chromatography, etc. The content analysis methods of diosmin include high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), RP-HPLC, HPLC-ESI-MS / MS, HPLC-DAD, which have high sensitivity and good accuracy, but these detection methods require the use of expensive instruments and equipment, high detection costs, long time consumption, and are not suitable for rapid on-site detection. Compared with instrumental analysis, immunoassay has the advantages of being fast, simple, real-time, easy to perform on-site detection, simple sample pretreatment, high sensitivity, strong selectivity, and suitable for high-throughput analysis, and can also significantly reduce the detection cost. Summary of the invention

[0006] Based on the above purpose, the purpose of the present application is to provide an antigen for detecting diosmin and an antibody obtained by the antigen, so that diosmin in a sample to be tested can be quickly detected by immunoassay, which has the advantages of being fast, simple, real-time, easy to perform on-site detection, simple sample pretreatment, high sensitivity, strong selectivity, and suitable for high-throughput analysis, and can also significantly reduce the detection cost.

[0007] To this end, the first technical solution of the present application discloses a diosmin antigen having a general structural formula as shown in A1 or A2:

[0008]

[0009] Among them, Protein is a carrier protein;

[0010] In the formula A1 shown, n is an integer of 1-6;

[0011] In the formula A2, a is an integer of 1-6, b is an integer of 0-6, and X is one of an aromatic ring and an aromatic heterocyclic compound.

[0012] Preferably, the carrier protein is one of bovine serum albumin, ovalbumin and hemocyanin.

[0013] The second technical solution of the present application discloses a method for preparing the above-mentioned diosmin antigen, which comprises the following steps:

[0014] S1. reacting dioscorea lignin with an ester-containing compound to obtain intermediate-1;

[0015] S2. hydrolyzing the intermediate-1 with a base to obtain the diosmin hapten;

[0016] S3. Diosmin hapten is subjected to coupling reaction with N-hydroxysuccinimide and a condensing agent to obtain intermediate-2;

[0017] S4. Intermediate-2 and carrier protein are coupled to obtain diosmin antigen;

[0018] Wherein, the dioscorea lignin has the structural formula shown in B:

[0019]

[0020] The ester group-containing compound has a structural formula as shown in C1 or C2:

[0021] Y-(CH2) n -COO-(CH2) m -CH3 (C1);

[0022] Y-(CH2) a -X-(CH2) b -COO-(CH2) m -CH3 (C2);

[0023] Wherein, X is one of an aromatic ring and an aromatic heterocyclic compound, Y is one of a Cl, Br and I group, n is an integer of 0-6, m is an integer of 0-6, a is an integer of 1-6, and b is an integer of 0-6;

[0024] The intermediate-1 has the structural formula shown in D1 or D2:

[0025]

[0026] The diosmin hapten has a structural formula as shown in E1 or E2:

[0027]

[0028] The intermediate-2 has the structural formula shown in F1 or F2:

[0029]

[0030] Furthermore, in the reaction described in S1, the molar ratio of dioscorea lignin to the ester-containing compound is 1:(0.1-10), the reaction temperature is 0-100°C, and the reaction time is 6-48h.

[0031] Furthermore, the coupling reaction described in S3 uses dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensation agent, wherein the molar ratio of diosmin hapten, N-hydroxysuccinimide and condensation agent is 1:(1-5):(1-5); the reaction temperature is 0-50°C, and the reaction time is 4-24h.

[0032] Furthermore, in the coupling reaction described in S4, the molar ratio of intermediate-2 to carrier protein is 1:(50-30), the reaction temperature is 0-50°C, the reaction time is 8-36h, and the pH is 5-9.

[0033] And, the diosmin antigen prepared according to the above preparation method has the structural formula shown as A1 or A2.

[0034] The third technical solution of the present application discloses an antibody obtained by using the above-mentioned diosmin antigen as an immunogen.

[0035] The fourth technical solution of the present application discloses the use of the above-mentioned diosmin antibody in the detection of diosmin.

[0036] The fifth technical solution of the present application discloses the use of the above-mentioned diosmin antibody in the preparation of a kit for detecting diosmin.

[0037] The beneficial effects of the present application are as follows: the present application prepares a diosmin antigen, obtains a diosmin antibody through the antigen, and then uses the antigen and the antibody to detect diosmin in the environment. Compared with the prior art, the present application can conveniently and quickly obtain a diosmin antigen, and the synthesis steps are simple and clear, the synthesis cost is low, and the effect is good. By immunizing with the diosmin antigen, the diosmin antibody with good specificity and high sensitivity can be obtained, which can be used for simple, convenient and rapid detection of diosmin, thereby reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Attached Figure 1 This is a flow chart of the synthesis of diosmin antigen in Example 1. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific examples. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following examples.

[0040] The first embodiment of the present application discloses a diosmin antigen having a general structural formula as shown in A1 or A2:

[0041]

[0042] Among them, Protein is a carrier protein;

[0043] In the formula A1 shown, n is an integer of 1-6;

[0044] In the formula A2, a is an integer of 1-6, b is an integer of 0-6, and X is one of an aromatic ring and an aromatic heterocyclic compound.

[0045] The carrier protein is one of bovine serum albumin, ovalbumin and hemocyanin.

[0046] The diosmin antigen described in this embodiment can be used as an immunogen to prepare diosmin antibodies, which are used for the detection of diosmin in the environment or in medicines and foods.

[0047] The second embodiment of the present application discloses a method for preparing the above-mentioned diosmin antigen, which comprises the following steps:

[0048] S1. reacting diosmetin with an ester-containing compound to obtain intermediate-1;

[0049] Wherein, the dioscorea lignin has the structural formula shown in B:

[0050]

[0051] The ester group-containing compound has a structural formula as shown in C1 or C2:

[0052] Y-(CH2) n -COO-(CH2) m -CH3 (C1);

[0053] Y-(CH2) a -X-(CH2) b -COO-(CH2) m -CH3 (C2);

[0054] Wherein, X is one of an aromatic ring and an aromatic heterocyclic compound, Y is one of Cl, Br and I groups, n is an integer of 0-6, m is an integer of 0-6, a is an integer of 1-6, and b is an integer of 0-6.

[0055] The compound represented by C1 can be selected from at least one of methyl chloroformate, ethyl chloroformate, ethyl chloroacetate, butyl chloroacetate, methyl chloropropionate, methyl chlorobutyrate, ethyl chlorobutyrate, propyl chlorobutyrate, methyl bromoacetate, ethyl bromoacetate, butyl bromoacetate, methyl bromobutyrate, ethyl bromobutyrate, butyl bromobutyrate, ethyl iodoacetate, methyl iodobutyrate, ethyl iodobutyrate, etc.; the compound represented by C2 can be selected from at least one of methyl p-chlorobenzoate, ethyl p-chlorophenylacetate, ethyl o-chlorophenylacetate, m-chlorophenylacetate, methyl p-bromophenylacetate, ethyl p-bromophenylacetate, ethyl p-bromophenylpropionate, methyl p-bromomethylbenzoate, ethyl p-bromomethylbenzoate, methyl m-bromomethylbenzoate, methyl m-bromobenzoate, ethyl m-bromobenzoate, methyl p-iodobenzoate, ethyl p-iodobenzoate, ethyl p-iodophenylacetate, methyl o-iodobenzoate, etc.

[0056] In the reaction, the molar ratio of dioscorea lignin to the ester-containing compound is 1:(0.1-10), the reaction temperature is 0-100° C., and the reaction time is 6-48 hours.

[0057] The reaction uses any one of pyridine, N,N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran as a solvent.

[0058] It should be noted that this reaction can also use K2CO3 as a catalyst to activate the free hydrogen at the hydroxyl position. In addition, Na2CO3 and the like can also be used as catalysts.

[0059] In this reaction, the synthesized intermediate-1 has the structural formula shown in D1 or D2:

[0060]

[0061]

[0062] S2. The intermediate-1 is subjected to a hydrolysis reaction with a base to obtain the diosmin hapten.

[0063] In this reaction, the base can be LiOH, NaOH, etc.

[0064] The diosmin hapten synthesized in the reaction has the structural formula shown in E1 or E2:

[0065]

[0066] S3. The diosmin hapten reacts with N-hydroxysuccinimide and a condensing agent through coupling reaction to obtain intermediate-2.

[0067] In the reaction, dicyclohexylcarbodiimide (DCC) or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is used as a condensation agent, wherein the molar ratio of diosmin hapten, N-hydroxysuccinimide and the condensation agent is 1:(1-5):(1-5); the reaction temperature is 0-50°C, and the reaction time is 4-24h.

[0068] In this reaction, the synthesized intermediate-2 has the structural formula shown in F1 or F2:

[0069]

[0070]

[0071] S4. The intermediate-2 is coupled with the carrier protein to obtain the diosmin antigen.

[0072] In the reaction, the molar ratio of intermediate-2 to carrier protein is 1:(50-30), the reaction temperature is 0-50°C, the reaction time is 8-36h, and the pH is 5-9.

[0073] In the reaction, the carrier protein is preferably one of bovine serum albumin (BSA), ovalbumin (OVA), and hemocyanin (KLH). The carrier protein participates in the reaction in the form of a solution. The solution is obtained by adding the carrier protein to a buffer solution. The buffer solution is selected from at least one of a carbonate buffer, a phosphate buffer, a borate buffer, and a 4-hydroxyethylpiperazineethanesulfonic acid buffer. The pH value of the buffer can be 7.4; and the reaction pH can be controlled to be 5-9 during the reaction.

[0074] The diosmin antigen obtained by the reaction has the structural formula shown in A1 or A2.

[0075]

[0076] It is understood that after the S4 reaction obtains the diosmin antigen, the reaction system is dialyzed to obtain a pure diosmin antigen compound. The preferred dialysis method is to use a phosphate buffer solution with a pH value of 4 to 10 and a concentration of 0.01 to 0.2 mol / L for dialysis.

[0077] It can be understood that in the reaction steps described in S1-S3, the collection of reaction products after each reaction step is also included, such as centrifugation, filtration, drying, etc. of the reaction products, which are conventional practices in the art and do not exceed the conventional knowledge of technicians in this field, and this application does not impose any restrictions on this.

[0078] The third embodiment of the present application discloses an antibody prepared using the diosmin antigen prepared by the preparation method of the first embodiment or the second embodiment. It is understood that the method of obtaining an antibody from an antigen is a conventional method in the art, which does not exceed the conventional cognition of those skilled in the art. The present application specifically adopts a method of using mice as experimental animals, injecting the diosmin antigen described in the present application as an immunogen to carry out an immune reaction, thereby obtaining a diosmin antibody, but this does not serve as a limitation to the present application of obtaining a diosmin antibody through a diosmin antigen.

[0079] The diosmin antibody obtained by using the diosmin antigen of the present application has the function of specifically recognizing diosmin in the environment, medicines, or foods, and can effectively detect diosmin.

[0080] It is understandable that the diosmin antigen and the antibody obtained from the antigen of the present application can also be prepared into an immunoassay kit for detecting diosmin in the environment, medicines, and foods, such as an enzyme-linked immunosorbent assay kit, a luminescent immunoassay kit, or an immunoaffinity chromatography column.

[0081] The detection effect of the diosmin antigen described in the present application and the diosmin antibody obtained by using the antigen on diosmin will be described below in conjunction with specific examples.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0083] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0084] Ovalbumin was purchased from Shanghai Shenggong Biotechnology Co., Ltd., bovine serum albumin and horseradish peroxidase-labeled goat anti-mouse immunoglobulin IgG-HRP were purchased from Aladdin Biochemical Technology Co., Ltd., diosmin and diosmin were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and DMF was purchased from Beijing Bailingwei Technology Co., Ltd.

[0085] Example 1 Preparation of diosmin-ovalbumin (diosmin-OVA) antigen, the reaction process is as follows Figure 1 shown.

[0086] S1. In a 50 mL three-necked flask, add 300 mg of geraniol, 205 mg of ethyl 4-bromobutyrate and 56 mg of potassium carbonate, then add 10 mL of DMF (solvent: N,N-dimethylformamide), react at room temperature for 18 h, add 20 mL of saturated sodium chloride, and extract with ethyl acetate (3×50 mL); dry with anhydrous magnesium sulfate, filter and spin dry.

[0087] Add 100 mg LiOH, then add 5 mL 80% methanol water, react at room temperature for 1 hour, remove methanol by rotary evaporation, adjust pH to 3, extract and separate the organic phase by adding ethyl acetate, dry with anhydrous magnesium sulfate, filter and rotary dry, and obtain the product by column chromatography.

[0088] The product was 1 H NMR, 13 C NMR and mass spectrometry confirmed that the product was the diosmin hapten shown in formula I, i.e., diosmin hapten (E1).

[0089]

[0090] in, 1 H NMR (400MHz, DMSO) δ12.92(s,1H),12.11(s,1H),9.43(s,1H),7.57(d,J=8.6Hz,1H),7.46(s,1H),7.10(d,J=8.6Hz, 1H),6.81(s,1H),6.76(s,1H),6.37(s,1H),4.12(t,J=6.2Hz,2H),3.88(s,3H),2.40(t,J=7.2Hz,2H),1.97(m,2H);

[0091] 13 C NMR(101MHz,DMSO)δ182.32,174.44,164.88,164.35,161.67,157.74,151.74,147.30,12 3.42,119.28,113.61,112.61,105.20,104.19,98.85,93.50,68.08,56.28,30.45,24.44.

[0092] S2. Weigh the diosmin hapten (11.5 mg), N-hydroxysuccinimide (5.13 mg), and DCC (N,N'-dicyclohexylcarboximide) (6.7 mg) obtained in S1, dissolve them in 1 mL of anhydrous DMF (solvent: N,N-dimethylformamide), stir and react overnight at room temperature 25°C, centrifuge the reaction solution at 8000 rpm for 5 minutes, and take the supernatant to obtain the compound of formula II (Intermediate-2, F1);

[0093]

[0094] It can be understood that in this step, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride can be used as a condensation agent and water can be used as a solvent for dissolution. When water is used as the solvent, the centrifugal separation step can be omitted.

[0095] S3. The intermediate-2 obtained in S2 is slowly added dropwise to a carrier protein OVA solution (the carrier protein solution is prepared by dissolving 43.7 mg OVA (ovalbumin) in 10 mL phosphate buffer (PBS) having a pH value of 7.4 and mixing evenly), the molar ratio of Formula II to the carrier protein OVA is 1:30, and the mixture is stirred at 4° C. overnight to obtain a reaction solution containing diosmin-OVA antigen;

[0096] The PBS solution was prepared as follows: NaCl, KH2PO4 and Na2HPO4·12H2O were dissolved in water at a mass ratio of 8.0:0.2:2.96, and the volume was adjusted to 1 L with water.

[0097] S4. The reaction solution obtained in S3 was dialyzed for three days with a PBS solution having a pH value of 7.4 and a concentration of 0.01 mol / L. The dialyzed reaction product solution (diosmin-OVA) was diluted to a 1 mg / mL solution and frozen at -80°C for later use. The function of dialysis is to remove unreacted diosmin and other small molecules to obtain the conjugate shown in formula III-1, i.e., diosmin antigen.

[0098]

[0099] Example 2 Preparation of diosmin-bovine serum albumin (diosmin-BSA) antigen.

[0100] Wherein, S1-S2 are as shown in S1-S2 in Example 1, to obtain Formula II (Intermediate-2, F1);

[0101] S3. Formula II was slowly added dropwise to a carrier protein solution (the carrier protein solution was 65.56 mg BSA dissolved in 10 mL phosphate buffer (PBS) with a pH value of 7.4), the molar ratio of the compound of Formula II to the carrier protein was 1:30, and stirred at 4 ° C overnight to obtain a reaction solution containing diosmin-BSA antigen;

[0102] The PBS solution was prepared as follows: NaCl, KH2PO4 and Na2HPO4·12H2O were dissolved in water at a mass ratio of 8.0:0.2:2.96, and the volume was adjusted to 1 L with water.

[0103] S4. The reaction solution was dialyzed for three days with a PBS solution having a pH value of 7.4 and a concentration of 0.01 mol / L. The dialyzed reaction product solution (diosmin-BSA) was diluted to a 1 mg / mL solution and stored at -80°C for later use. The function of dialysis is to remove unreacted diosmin hapten and other small molecules to obtain the conjugate shown in formula III-2, i.e., diosmin-BSA antigen.

[0104]

[0105] Example 3 Application of diosmin-bovine serum albumin (diosmin-BSA) antigen

[0106] Experiment 1 Preparation of diosmin-BSA antibody

[0107] (1) 8-10 week old Balb / c mice were used as experimental animals.

[0108] (2) Basic immunization: The diluted diosmin-BSA antigen solution (concentration of 1 mg / mL) obtained in Example 2 was filtered through a sterile filter, and an equal volume of Freund's complete adjuvant was added. The mixture was stirred and emulsified with a magnetic stirrer until it did not spread when dropped into water. The emulsified complete antigen was injected into Balb / c mice at multiple points in the peritoneal cavity and the back subcutaneously, with an injection dose of 0.1 mg of emulsified antigen per mouse.

[0109] (3) Booster immunization: 2 weeks after the basic immunization, take 1 mL of the diluted diosmin-BSA antigen solution, then add 1 mL of Freund's incomplete adjuvant, and stir thoroughly with a magnetic stirrer until it does not spread when dropped into water. The emulsified antigen is injected into Balb / c mice at multiple points in the abdominal cavity and back subcutaneously, with an injection dose of 0.1 mg of emulsified diluted antigen per mouse (8-week-old Balb / C mice weigh about 23-25 ​​g).

[0110] The booster immunization was performed every 15 days. Starting from the third booster immunization, blood was collected from the mouse eye sockets on the 3rd to 5th day after each immunization to determine the antibody titer. The original 1mg / mL diosmin-OVA was diluted 500 times. When the titer was greater than 1:8000 (the titer was defined as the dilution multiple of the serum when the zero-hole color value was 1), the eyeball was enucleated and blood was collected. After the blood was allowed to stand at room temperature for 1 hour, it was allowed to stand in a refrigerator at 4°C for 2 hours, and then centrifuged at 8000r / min for 5 minutes in a centrifuge to separate the antiserum, that is, diosmin-BSA antibody. This antibody was used in the following experiments.

[0111] Experiment 2 Antibody effect detection

[0112] The various buffers used in the following experiments are as follows:

[0113] (1) Coating buffer: 0.05 M carbonate buffer, pH 9.6;

[0114] (2) Phosphate buffer (PBS) (pH 7.4): weigh 4.0 g NaCl, 0.1 g KH2PO4, and 1.48 g Na2HPO4·12H2O and dilute to 500 mL with distilled water to obtain a phosphate buffer solution with a concentration of 0.01 M and a pH of 7.4.

[0115] (3) Sample diluent PBSTG: 0.5 mL Tween 20, 0.5 g gelatin, and 500 mL 0.1 M PBS buffer with a pH of 7.4 were mixed;

[0116] (4) Stop buffer: 2 M sulfuric acid aqueous solution;

[0117] (5) Washing solution: composed of NaCl, KH2PO4, Na2HPO4·12H2O, Tween-20 and water; the concentration of NaCl in the washing solution is 8.0 g / L, the concentration of KH2PO4 in the washing solution is 0.2 g / L, the concentration of Na2HPO4·12H2O in the washing solution is 2.96 g / L, and the volume percentage of Tween-20 in the washing solution is 1:1000.

[0118] (I) Antibody inhibition experiment

[0119] 1. Preparation of diosmin-OVA coated antigen solution

[0120] After the diluted 1 mg / mL diosmin-OVA antigen prepared in Example 1 was completely thawed, it was gradiently diluted with coating buffer at 1:1000, 1:2000, 1:4000, and 1:8000 to obtain coating antigen solutions of diosmin-OVA with different concentrations.

[0121] 2. Preparation of Diosmin Standard Solution

[0122] (1) Weigh 10 mg of diosmin standard sample and fully dissolve it in 10 mL of dimethyl sulfoxide to obtain a 1.0 mg / mL diosmin standard solution;

[0123] (2) The 1.0 mg / mL diosmin standard solution prepared in step (1) above was prepared with sample diluent to obtain a diosmin standard solution having a concentration of 1000 ng / mL.

[0124] 3. Preparation of diosmin-BSA antiserum dilution

[0125] The diosmin-BSA antibody prepared in step 1 was diluted with the sample diluent at a gradient of 1:2000, 1:4000, 1:8000, and 1:16000 to obtain a diosmin-BSA antiserum dilution.

[0126] 4. Antigen and Antibody Chessboard Experiment

[0127] Coating: Add 100 μL of the diosmin-OVA coating antigen solution prepared in step 1 to each well of a 96-well ELISA plate, coat at 37° C. for 3 hours, and wash four times with washing solution.

[0128] Blocking: Weigh 5g skim milk powder and fully dissolve it in 100mL sample diluent to obtain 5% blocking solution. Add 150μL blocking solution to each well of a 96-well ELISA plate, block in a 37℃ wet box for 1h, discard the blocking solution, and wash 4 times.

[0129] Competition: Add 50 μL of sample diluent to each well of the zero well, and add 50 μL of the diosmin standard solution prepared in step 2 to each well of the inhibition well. Add the diosmin-BSA antiserum dilution obtained in step 3 above (from 2×10 3 times to 16×10 3 The plate was added with 50 μL / well of the ELISA plate, placed in a humidified box at 37°C for 30 min, and washed 4 times.

[0130] Add enzyme-labeled secondary antibody: dilute goat anti-mouse enzyme-labeled secondary antibody (0.1 mg / mL) 1000 times in 0.01 M PBSTG, pH 7.4. Add 100 μL to each well, incubate in a humidified box at 37°C for 30 min, and wash the plate 4 times.

[0131] Color development: Add single-component TMB color development solution to the ELISA plate, 100 μL per well. Color development for 15 minutes in the dark.

[0132] Stop: Add 50 μL of stop buffer to each well and measure the OD value of each well at 450 nm using an ELISA reader.

[0133] The titer was defined as the serum dilution factor at which the OD value of the zero well was 1.

[0134] The results are shown in Table 1.

[0135] Table 1. Serum titer detection of anti-diosmin mice (TMB colorimetric solution colorimetric 10 min at room temperature, 1000 ng standard sample inhibition)

[0136]

[0137]

[0138] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0139] The results show that the diosmin-BSA prepared in Example 1 can be used as an immunogen to prepare antibodies for detecting diosmin.

[0140] It should be noted that for the diosmin antigen A2 described in the present application, since antibodies can only be produced for macromolecules that can participate in immune responses, based on the exposed recognition sites, recognition site antibodies at different sites are produced, and conventionally every 5-6 carbon atom groups can form a recognition cluster. Diosmin is the characteristic structure of diosmin. In the present application, A1-A2 all contain the structure of diosmin, which is only different in the spacer arms. The purpose is to expose the structure of diosmin on the protein surface, so that the generated antibodies can recognize diosmin. Therefore, A1 / A2 is essentially the same, and the difference structure of A1 / A2 only plays a bridging role. Therefore, when A1 can produce an effect, those skilled in the art can obtain the result that A2 can also produce the same technical effect.

[0141] Finally, it should be noted that the above implementation cases are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred implementation cases, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A diosmin antigen having the general structural formula shown in A1 or A2: in, Protein is a carrier protein; In the formula A1 shown, n is an integer of 1-6; In the formula A2, a is an integer of 1-6, b is an integer of 0-6, and X is one of an aromatic ring and an aromatic heterocyclic compound.

2. The diosmin antigen according to claim 1, characterized in that The carrier protein is one of bovine serum albumin, ovalbumin and hemocyanin.

3. A method for preparing the diosmin antigen as claimed in claim 1 or 2, comprising the following steps: S1. reacting diosmetin with an ester-containing compound to obtain intermediate-1; S2. hydrolyzing the intermediate-1 with a base to obtain the diosmin hapten; S3. The diosmin hapten reacts with N-hydroxysuccinimide and a condensing agent to obtain intermediate-2; S4. Intermediate-2 and carrier protein are coupled to obtain diosmin antigen; in, The diosmetin has a structural formula as shown in B: The ester group-containing compound has a structural formula as shown in C1 or C2: Y-(CH2) n -COO-(CH2) m ―CH3 (C1); Y-(CH2) a -X-(CH2) b -COO-(CH2) m -CH3 (C2); Wherein, X is one of an aromatic ring and an aromatic heterocyclic compound, Y is one of Cl, Br and I groups, n is an integer of 0-6, m is an integer of 0-6, a is an integer of 1-6, and b is an integer of 0-6; the intermediate-1 has the structural formula shown in D1 or D2: The diosmin hapten has a structural formula as shown in E1 or E2: The intermediate-2 has the structural formula shown in F1 or F2:

4. The preparation method according to claim 3, characterized in that: In the reaction described in S1, the molar ratio of dioscorea lignin to the ester-containing compound is 1:(0.1-10), the reaction temperature is 0-100°C, and the reaction time is 6-48h.

5. The preparation method according to claim 3, characterized in that: The coupling reaction in S3 uses dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride as a condensing agent, wherein the molar ratio of diosmin hapten, N-hydroxysuccinimide and the condensing agent is 1:(1-5): (1-5); the reaction temperature is 0-50°C, and the reaction time is 4-24h.

6. The preparation method according to claim 3, characterized in that: In the coupling reaction described in S4, the molar ratio of intermediate-2 to carrier protein is 1:(50-30), the reaction temperature is 0-50°C, the reaction time is 8-36h, and the pH is 5-9.

7. The diosmin antigen prepared according to any one of the preparation methods according to claims 3-6, having the structural formula shown in claim 1, A1 or A2.

8. A diosmin antibody obtained by using the diosmin antigen according to any one of claims 1 to 2 or claim 7 as an immunogen.

9. Use of the diosmin antibody according to claim 8 in detecting diosmin.

10. Use of the diosmin antibody according to claim 8 in preparing a kit for detecting diosmin.

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