Amurenone antigen as well as preparation method and application thereof

Abstract

The invention relates to an obakunone antigen as well as a preparation method and application thereof, and the preparation method specifically comprises the following steps: firstly, reacting obakunone with a compound B containing amino and carboxyl at the same time to generate a compound C; then, in the presence of dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the compound C and N-hydroxysuccinimide are subjected to a coupling reaction, and a compound D is obtained; and finally, synthesizing the obakunone antigen through coupling reaction with carrier protein. The obakunone antigen can be used for preparing an enzyme linked immunosorbent assay kit, a luminescence immunoassay kit or an immunoaffinity chromatographic column for detecting obakunone in a sample. The method has the advantages that the obakunone antigen can be efficiently and conveniently synthesized, the synthesis steps are simple and clear, the cost is low, and the effect is remarkable. In addition, the antibody obtained by using the obakunone antigen has good specificity and lower minimum detection limit.

Description

Technical Field

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

[0002] Obacunone is commonly known as Obacunone; its chemical name is Obacunone; its trade name is Obacunone; its CAS registration number is 751-03-1; its molecular formula is C26H30O7; and its relative molecular weight is 454.51. Its chemical structure is as follows:

[0003]

[0004] Obacunone belongs to the natural limonoid compound, mainly found in Rutaceae plants (such as Phellodendron amurense and citrus fruits). It has a variety of significant physiological functions and pharmacological activities, including: inhibiting inflammation-related signaling pathways (such as NF-κB, MAPK), reducing the release of pro-inflammatory factors (such as TNF-α, IL-6), and having potential therapeutic effects on chronic inflammatory diseases (such as arthritis and enteritis); protecting cells from oxidative damage, delaying the aging process, and combating oxidative stress-related diseases (such as neurodegenerative diseases) by scavenging free radicals (ROS) and enhancing the activity of antioxidant enzymes (such as SOD, GSH-Px); inducing tumor cell apoptosis (such as by activating Caspase-3 / 9), inhibiting cancer cell proliferation (such as breast cancer and colon cancer), and blocking tumor metastasis (by inhibiting MMPs enzymes); it has inhibitory effects on a variety of bacteria (such as Staphylococcus aureus) and fungi (such as Candida albicans), and can also interfere with viral replication (such as influenza virus, HSV-1), enhancing the body's ability to resist infection.

[0005] At present, the main methods for extracting chloroquine include traditional solvent extraction, ultrasonic or microwave-assisted extraction, supercritical CO2 extraction, etc. Its content analysis methods include high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), RP-HPLC, HPLC-ESI-MS / MS, and HPLC-DAD, which have high sensitivity and good accuracy. However, the above traditional detection methods have obvious limitations: on the one hand, they need to rely on high-value precision instruments and equipment, resulting in high costs for single detection; on the other hand, the analysis process is complicated and lengthy, making it difficult to meet the timeliness requirements of on-site rapid detection. In comparison, immunoassay technology exhibits the following outstanding advantages: ease of operation, on-site applicability, method stability, superior performance, and throughput advantages. Summary of the Invention

[0006] In order to solve the above technical defects, the present application provides a genotype antigen and its preparation method and application. The method can obtain the genotype antigen conveniently and quickly, and has broad application prospects in rapid immunoassay of genotype antigen.

[0007] To this end, the first technical solution of the present application discloses a bevacizumab antigen having the general structural formula shown in A:

[0008]

[0009] Wherein, X is one of O, S, CH2, and NH groups, and n is an integer of 0-6; Protein represents a carrier protein, and the carrier protein is selected from any one of bovine serum albumin and ovalbumin.

[0010] The second technical solution of the present application discloses a method for preparing the above-mentioned obacillus antigen, comprising the following steps:

[0011] S1. reacting chloroquine with compound B to obtain compound C;

[0012] S2. Compound C is subjected to a coupling reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain Compound D;

[0013] S3. Compound D is coupled with a carrier protein to obtain the chloroquine antigen A;

[0014] Among them, compound B is a compound containing both amino group and carboxylic acid, and its general structural formula is:

[0015] H2N-X-(CH2)n-COOH (B);

[0016] The general structural formula of compound C is:

[0017]

[0018] The general structural formula of compound D is:

[0019]

[0020] In formula B, C, and D, X is one of O, S, CH2, and NH groups, and n is an integer from 0 to 6;

[0021] The carrier protein is selected from at least one of bovine serum albumin and ovalbumin.

[0022] Preferably, the molar ratio of compound B to benzyl alcohol in S1 is (0.1-10):1; wherein compound B is preferably at least one of carboxymethyloxyamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminovaleric acid and aminocaproic acid.

[0023] Preferably, the reaction temperature in S1 is 0-100° C., and the reaction time is 6-48 h.

[0024] Preferably, the solvent in the S1 reaction is selected from at least one of pyridine, N,N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

[0025] Preferably, the molar ratio of compound C, N-hydroxysuccinimide and dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in S2 is 1:(1-5):(1-5).

[0026] Preferably, the coupling reaction temperature in S2 is 0-50° C., and the reaction time is 4-24 h.

[0027] Preferably, the molar ratio of compound D to carrier protein in S3 is (5-30):1, the coupling reaction temperature is 0-50°C, the reaction time is 8-36h, and the pH of the reaction system is 5-9.

[0028] Furthermore, compound D is subjected to a coupling reaction in a solution of the carrier protein, wherein the solution of the carrier protein is obtained by adding the carrier protein to a buffer solution, wherein 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, and the pH value of the buffer can be 7.4.

[0029] Preferably, after the coupling reaction in steps S2 and S3, the method further includes dialyzing the reaction system of the coupling reaction; in the dialysis step, the dialysate used is a phosphate buffer solution with a pH value of 4 to 10 and a concentration of 0.01 to 0.2 mol / L.

[0030] Preferably, the bacterin and nomilin antigens are conjugates formed by connecting bacterin and a carrier protein via an amide bond; the amide bond is formed by the carboxyl group on formula C and the amino group on the carrier protein via an active ester.

[0031] And, the Houttuynia cordata antigen obtained according to the above preparation method.

[0032] and antibodies prepared based on the above-mentioned Houttuynia cordata antigen.

[0033] The third technical solution of the present application discloses the use of the above-mentioned benzophenone antigen or antibody in detecting benzophenone in medicines and foods.

[0034] Furthermore, the said procakone antigen or antibody is prepared into an enzyme-linked immunosorbent assay kit for Nomilin, a luminescent immunoassay kit for procakone or an immunochromatographic test strip for detecting procakone in the sample to be tested.

[0035] The beneficial effects of this application are as follows: The method for preparing a bacterium ketone antigen disclosed in this application can conveniently and quickly obtain the bacterium ketone antigen, with simple and clear synthesis steps, low synthesis cost, and good efficacy. Antibodies obtained by immunization with the bacterium ketone and nomilin antigens prepared by the method of the present invention have good specificity and a low minimum detection limit. The method for preparing a bacterium ketone antigen of the present invention and the bacterium ketone obtained by the method will have broad prospects in the application of rapid immunoassays. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The synthetic route of the phellodendron antigen is shown below;

[0037] Figure 2 This is the standard curve for the established indirect ELISA method for phellodendron. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to specific examples. This embodiment is carried out based on the technology of the present invention, and detailed implementation methods and specific operation processes are now given to illustrate that the present invention is creative, but the scope of protection of the present invention is not limited to the following examples.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin, and ovalbumin were purchased from Sigma. Goat anti-mouse IgG-HRP was purchased from Jackson. Other conventional reagents, such as carboxymethyloxyamine hemihydrochloride and chloroquine, were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0041] Example 1. Preparation of Obacterial-Ovalbumin (OVA) Antigen

[0042] Synthesis route diagram Figure 1 shown.

[0043] 1) Synthesis of the compound represented by formula I

[0044] In a 50 mL flask, add 0.3 g of chloroquine and 0.2 g of carboxymethyloxyamine hemihydrochloride (one of the compounds B, where x is O and n = 1), then add 15 mL of pyridine, heat to 100 degrees, react for 24 hours, and then cool to room temperature. Pour the reaction solution into 100 mL of water, adjust the pH to 3 with concentrated hydrochloric acid, extract with ethyl acetate (3×50 mL), dry over anhydrous sodium sulfate, and desolventize by rotary column chromatography to obtain 0.2 g of the product with a yield of 56%.

[0045] 1 H NMR (400MHz, DMSO) δ7.73(s,1H,H-23),7.66(s,1H,H-21),6.73(d,J=11.8Hz,1H,H-1),6. 50(s,1H,H-22),5.81(d,J=11.7Hz,1H,H-2),5.45(s,1H,H-17),4.22(s,2H,H-24),3.68(s ,1H,H-15),2.21(m,2H,H-6),1.80(m,2H,H-11),1.67(m,2H,H-12),1.44(s,3H,H-26),1.3 7(s,3H,H-27),1.37(s,1H,H-9),1.28(s,3H,H-19),1.12(s,3H,H-25),1.07(s,3H,H-18).

[0046] 13 C NMR(101MHz,DMSO)δ167.37(COOH),167.32(C-16),159.89(C-3),159.50(C-7,C-1),143.78(C-23),14 2.02(C-21),121.95(C-2),120.79(C-20),110.72(C-22),84.89(C-4),78.23(C-17),72.71(C-24),65. 70(C-14),55.42(C-15),53.81(C-5),49.59(C-10),45.94(C-9),43.17(C-13),37.71(C-8),33.11(C- 27),32.45(C-26),27.23(C-12),24.05(C-19),21.63(C-11),19.66(C-18),17.02(C-6),16.44(C-25).

[0047] ESI-MS m / z 528.30 [M+H] + ,550.20[M+Na] + .

[0048] Product 1 H-NMR, 13 C-NMR and mass spectrometry confirmed that it was the hapten compound of the formula I (one of the compounds C, wherein x is O and n=1).

[0049]

[0050] 2) Coupling reaction

[0051] Method (1): The hapten of formula I (0.036 mmol), NHS (0.047 mmol), and DCC (0.040 mmol) obtained in step 1) were weighed and dissolved in 1 mL of anhydrous DMF. After stirring at room temperature (25°C) for 6 hours, the reaction solution was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected to obtain a compound of formula II (one of compound D: wherein x is O and n=1);

[0052] Method (2): The hapten of formula I (0.036 mmol), NHS (0.047 mmol), and EDC (0.040 mmol) obtained in step 1) were weighed and dissolved in 1 mL of water. The mixture was stirred at room temperature (25°C) for 6 hours to obtain a compound of formula II (one of compound D: wherein x is O and n=1).

[0053]

[0054] 3) The compound of Formula II obtained in step 2) was slowly added dropwise to a carrier protein OVA (ovalbumin) solution (the carrier protein solution was prepared by dissolving 105 mg of OVA in 10 mL of phosphate buffer (PBS) at a pH of 7.4 and mixing uniformly), with a molar ratio of the compound of Formula II to the carrier protein of 15:1, and stirred at 4°C overnight.

[0055] 4) Dialysis: The reaction solution from step 3) was dialyzed against PBS (pH 7.4, 0.01 mol / L) for three days. The dialyzed reaction product (obacterial ketone-OVA) was diluted to a 1 mg / mL solution and frozen at -40°C until use. Dialysis removed unreacted obacterial ketone hapten and other small molecules, yielding the conjugate of obacterial ketone and OVA represented by Formula III-1, also known as the obacterial ketone antigen represented by Formula A (wherein x is O, n=1, and protein is OVA).

[0056]

[0057] 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.

[0058] Example 2. Preparation of Houttuynia cordata-bovine serum albumin (Houttuynia cordata-BSA) antigen

[0059] 1) The synthesis and activation of the hapten represented by Formula I are the same as those in Example 1 and will not be described in detail here.

[0060] 2) The compound of formula II obtained in step 2) of Example 1 was slowly added dropwise to the carrier protein solution.

[0061] (The carrier protein solution is prepared by dissolving 157.5 mg of BSA (bovine serum albumin) in 10 mL of phosphate buffered saline (PBS) with a pH of 7.4), the molar ratio of the compound of formula II to the carrier protein is 15:1, and the mixture is stirred at 4°C overnight.

[0062] 3) Dialysis: The reaction solution from step 2) was dialyzed against PBS (pH 7.4, 0.01 mol / L) for three days. The dialyzed reaction product (BSA-BSA) was diluted to a 1 mg / mL solution and frozen at -40°C until use. Dialysis removed unreacted BSA hapten and other small molecules, yielding the BSA-BSA conjugate of Formula III-2, also known as the BSA antigen of Formula A (wherein x is O, n=1, and protein is BSA).

[0063]

[0064] 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.

[0065] Example 3: Application of the Houttuynia cordata-bovine serum albumin (Houttuynia cordata-BSA) antigen

[0066] 1. Preparation of antibodies using the flavonoid-bovine serum albumin (flavonoid-BSA) antigen

[0067] (1) 8-10 week old Balb / c mice were used as experimental animals.

[0068] (2) Primary Immunization: An equal volume of complete Freund's adjuvant (CFA) was added to the diluted H. sutsugamushi-BSA antigen solution (1 mg / mL) obtained in Example 2. The mixture was stirred thoroughly with a magnetic stirrer until emulsified and did not diffuse when dropped into water. The emulsified complete antigen was injected intraperitoneally and subcutaneously into the back of Bal b / c mice at multiple injection sites, with a dose of 0.1 mg of emulsified antigen per mouse.

[0069] (3) Booster Immunization: Two weeks after the primary immunization, take 1 mL of the diluted pyruvate-BSA antigen solution and add 1 mL of Freund's incomplete adjuvant. Stir thoroughly with a magnetic stirrer until the solution is emulsified and does not spread when dropped into water. Inject the emulsified antigen into Bal b / c mice intraperitoneally and subcutaneously at multiple points on the back. The injection dose for each mouse is 0.1 mg of the emulsified diluted antigen (8-week-old Bal b / c mice weigh approximately 23-25 g).

[0070] 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 1 mg / mL 1mg / mL 1% pyruvate-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 eyeballs were removed and blood was collected. After the blood was allowed to stand at room temperature for 1 hour, it was allowed to stand in a 4°C refrigerator for 2 hours, and then centrifuged at 8000 r / min for 5 minutes in a centrifuge to separate the serum to obtain the 1% pyruvate-BSA antibody.

[0071] (4) Cell fusion: Spleen cells were collected from mice after boosting immunization and fused with myeloma cells using PEG-2000 at a ratio of 10:1. After 10 days of cell culture in HAT medium (DMEM culture medium supplemented with 20% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin, and 1% HAT), the cell supernatant was tested by icELISA. Hybridoma cells with high titer and high inhibitory effect were screened, and then monoclonal cell lines were obtained by limiting dilution and amplified to ensure monoclonality. This experiment was performed in a clean bench.

[0072] (5) Ascites Collection: Paraffin is injected into the peritoneal cavity of mice. One week later, the cultured monoclonal hybridoma cells are intraperitoneally injected into the mice. After 7-10 days, the resulting ascites is collected and stored at -80°C.

[0073] 2. Antibody Effect Testing

[0074] The various buffers used in the following experiments are as follows:

[0075] (1) Coating buffer: 0.05 M carbonate buffer, pH 9.6;

[0076] (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.

[0077] (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;

[0078] (4) Citrate-phosphate buffer: composed of trisodium citrate, Na2HPO4 and water; the concentration of trisodium citrate in the citrate-phosphate buffer is 0.01 M, and the concentration of Na2HPO4 in the citrate-phosphate buffer is 0.03 M; the pH value of the citrate-phosphate buffer is 5.5;

[0079] (5) Substrate buffer: 20.0 mg of o-phenylenediamine (OPD) was dissolved in 10.0 mL of citrate-phosphate buffer, and then 4 μL of a 30% by volume aqueous solution of H2O2 was added. The citrate-phosphate buffer was as described in (4);

[0080] (6) Stop buffer: 2.0 M sulfuric acid aqueous solution;

[0081] (7) 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.

[0082] (1) Antibody inhibition experiment

[0083] 1. Preparation of Houttuynia cordata-OVA coated antigen solution

[0084] After the diluted 1 mg / mL pyruvate-OVA antigen prepared in Example 1 was completely thawed, it was gradiently diluted with coating buffer at 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, and 1:160000 to obtain coating antigen solutions of different concentrations of pyruvate-OVA.

[0085] 2. Preparation of Houttuynia cordata standard solution

[0086] (1) Weigh 10 mg of the standard sample of chloroquine and dissolve it in 10 mL of anhydrous methanol to obtain a 1.0 mg / mL chloroquine standard solution.

[0087] (2) The 1.0 mg / mL chloroquine standard solution prepared in step (1) was prepared with sample diluent to prepare a 1000 ng / mL chloroquine standard solution.

[0088] 3. Preparation of Houttuynia cordata monoclonal antibody (ascites)

[0089] The bactericidal monoclonal antibody (ascites) prepared in the above step 1 was serially diluted with the sample diluent at dilutions of 1:10,000, 1:20,000, 1:40,000, and 1:80,000 to obtain a bactericidal monoclonal antibody (ascites) dilution solution.

[0090] 4. Antigen and Antibody Checkerboard Experiment

[0091] Coating: Add 100 μL of the obacillus-OVA coating antigen solution prepared in step 1 to each well of a 96-well ELISA plate, incubate at 37°C for 3 hours, and wash four times with washing solution.

[0092] Competition: Add 50 μl of sample diluent to each well of zero well, and add 50 μl of the standard solution of chloroquine prepared in step 2 to each well of inhibition well. 3 times to 80×10 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.

[0093] Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson, catalog number 79556) (0.1 mg / mL) 1000-fold in 0.01 M PBSTG, pH 7.4. Add 100 μL to each well and incubate in a humidified chamber at 37°C for 30 min. Wash the plate four times.

[0094] Color development: Add 100 μl of substrate buffer to each well of the ELISA plate. Incubate in the dark for 15 minutes.

[0095] Stop: Add 50 μL of stop buffer to each well and measure the OD value of each well at 450 nm using a microplate reader.

[0096] The titer was defined as the serum dilution factor at which the OD value of the zero well was 1.

[0097] The results are shown in Table 1.

[0098] Table 1. Serum titer detection of anti-obacillus ketone mice (OPD color development at room temperature for 10 minutes, 1000ng standard sample inhibition)

[0099]

[0100] Note: I represents the inhibition well in the ELISA plate, and C represents the control well in the ELISA plate.

[0101] The results show that the hapten and antigen of bevacizumab ketone can be obtained through the above Examples 1 and 2, and the antibody for detecting bevacizumab ketone can be prepared through Example 3.

[0102] (2) Establishment of the standard curve of chloroquine

[0103] The prepared standard solution of chloroquine was diluted with sample diluent to the following concentrations: 12.500 ng / mL, 6.500 ng / mL, 3.125 ng / mL, 1.563 ng / mL, 0.781 ng / mL, 0.391 ng / mL,

[0104] 0.195ng / mL, 0.098ng / mL.

[0105] (1) Coating of the coating agent: dilute the above-prepared obacillus-OVA antigen at 1:80,000 and add it to the ELISA plate, 100 μL per well, and incubate at 37°C for 3 hours; pour off the solution in the ELISA plate, wash the plate 4 times with washing solution, and spin dry;

[0106] (2) Add the above-mentioned different concentrations of the standard solution of chloroquine to the ELISA plate in step (1) (experimental wells), 50 μL per well, and add 50 μL of sample diluent to the control wells without adding the standard solution of chloroquine;

[0107] (3) Add 50 μL of 1:10,000 dilution of chloroquine monoclonal antibody (ascites) to each well of the experimental wells and control wells; incubate at 37°C for 30 minutes; discard the solution in the ELISA plate, wash the plate four times with washing solution, and spin dry;

[0108] (4) Add 100 μL of IgG-HRP (Jackson, catalog number 79556) (0.1 mg / mL) diluted 1:1000 to each of the experimental and control wells and incubate at 37°C for 30 minutes. Wash the plate four times with washing solution, discard the solution from the plate, and spin dry.

[0109] (5) Add 100 μL of substrate buffer to the experimental wells and control wells, incubate at 37°C for 15 minutes, and then add 50 μL of 2.0 M sulfuric acid solution to each well to terminate the reaction;

[0110] (6) measuring the absorbance at 492 nm;

[0111] (7) Drawing a standard curve: Use different concentrations (ng / mL) of the standard solution of chloroquine as the X-axis and the absorbance ratio (B / B0×100%, where B is the average absorbance value of the chloroquine standard solution and B0 is the average absorbance value of the control well) as the Y-axis to draw a standard curve.

[0112] The experiment was repeated three times, and the average of the three experimental results was taken. The obtained standard curve is as follows Figure 2 The results show that its sensitivity (IC 50 ) was 0.44 ng / mL, and the detection range was 0.09 ng / mL-3.52 ng / mL. This indicated that the antibody obtained by immunizing mice with the monoclonal antibody (ascites) prepared in Example 1 as an antigen had a good effect.

[0113] (III) Antibody specificity detection

[0114] 1. Preparation of standard solution of chloroquine analogues

[0115] Preparation of standard samples of chloroquine analogs

[0116] Refer to the preparation method of the standard substance of malathione in step (1) to prepare standard samples of nomilin and limonin.

[0117] Use sample diluent to dilute the above-mentioned nomilin and limonin to the following concentrations: 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.50 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL, 3.91 ng / mL, and 1.95 ng / mL.

[0118] 2. Establish a standard curve and determine the inhibitory concentration IC 50 (The standard sample concentration value at which the inhibition rate reaches 50%).

[0119] The method for establishing the standard curve is the same as the method for establishing the above-mentioned standard curve of chloroquine.

[0120] Cross-reaction rate (%) = (Cyclohexene IC 50 ) / (Hydroxyproline analogous compounds IC 50 )×100%.

[0121] The experiment was repeated three times, and the average value of the three experimental results was taken. The results are shown in Table 2.

[0122] Table 2. Specificity detection of antibodies prepared from chloroquine-BSA

[0123] Analytes <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Houttuynia cordata 0.37 100 Nomilin 7.87 5.6 Limonin 31.85 1.4

[0124] The results showed that the cross-reactivity rate of the antibody prepared from the above-mentioned bactericidal ketone-BSA with its analogs nomilin and limonin was very low, indicating that the antibody prepared from the bactericidal ketone-BSA had good specificity for bactericidal ketone.

[0125] Those skilled in the art will appreciate that the embodiments described herein are intended to help readers understand the implementation methods of the present invention, and it should be understood that the scope of protection of the present invention is not limited to such specific descriptions and embodiments. Those skilled in the art can make various other specific variations and combinations based on the technical teachings disclosed in the present invention without departing from the essence of the present invention, and such variations and combinations are still within the scope of protection of the present invention.

Claims

1. A bacterium-containing antigen, characterized in that: It has the general structural formula shown in A: Wherein, X is one of O, S, CH2, and NH groups, and n is an integer of 0-6; Protein represents a carrier protein, and the carrier protein is selected from any one of bovine serum albumin and ovalbumin.

2. A method for preparing the Houttuynia cordata antigen according to claim 1, characterized in that: The steps include: S1. reacting chloroquine with compound B to obtain compound C; S2. Compound C is subjected to a coupling reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to obtain Compound D; S3. Compound D is coupled with a carrier protein to obtain the chloroquine antigen A; Among them, compound B is a compound containing both amino group and carboxylic acid, and its general structural formula is: H2N-X-(CH2)n-COOH (B); The general structural formula of compound C is: The general structural formula of compound D is: In formula B, C, and D, X is one of O, S, CH2, and NH groups, and n is an integer from 0 to 6; The carrier protein is selected from at least one of bovine serum albumin and ovalbumin.

3. The preparation method according to claim 2, characterized in that The compound B described in S1 is any one of carboxymethyloxyamine, hydrazinoacetic acid, aminoacetic acid, aminopropionic acid, aminobutyric acid, aminovaleric acid and aminocaproic acid.

4. The preparation method according to claim 2, characterized in that The molar ratio of compound B to chloroquine in S1 is (0.1-10):1; The reaction temperature is 0-100°C, and the reaction time is 6-48h.

5. The preparation method according to claim 2, characterized in that: The molar ratio of compound C, N-hydroxysuccinimide and dicyclohexylcarbodiimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in S2 is 1:(1~5):(1~5).

6. The preparation method according to claim 2, characterized in that: The coupling reaction temperature in S2 is 0-50° C., and the reaction time is 4-24 h.

7. The preparation method according to claim 2, characterized in that: The molar ratio of compound D to carrier protein in S3 is (5-30):1, the coupling reaction temperature is 0-50°C, the reaction time is 8-36h, and the pH of the reaction system is 5-9.

8. A benign protozotocin antigen prepared according to the preparation method according to any one of claims 2 to 7.

9. The antibody prepared according to the obacillus antigen according to claim 1 or 8.

10. Use of the bacterium compressa antigen according to claim 1 or 8 or the antibody according to claim 9 in detecting bacterium compressa in medicines and foods.

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