Hapten, complete antigen and antibody of aristolochic acid I and application of hapten, complete antigen and antibody in detection

Abstract

The invention belongs to the technical field of immunoaffinity chromatography, and particularly relates to a hapten, a complete antigen and an antibody of aristolochic acid I and application of the hapten, the complete antigen and the antibody to detection. 7-OH-aristolochic acid I reacts with succinic anhydride to obtain an aristolochic acid I hapten, the aristolochic acid I hapten is connected with carrier protein for animal immunization to prepare a corresponding artificial antibody, an immunoaffinity column for detecting aristolochic acid I is provided based on the artificial antibody, and the maximum binding capacity of the affinity column is about 2000ng; the method has the advantages of simple operation, high sensitivity, high speed, low cost and the like, can be repeatedly used for three times, has the recovery rate of not less than 90%, can be applied to rapid enrichment and recovery of aristolochic acid I in decoction pieces or Chinese patent medicines, and is suitable for screening and on-site monitoring of a large number of samples.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of immunoaffinity chromatography, and particularly relates to a hapten of aristolochic acid I, a complete antigen, an antibody, and application of preparation of an immunoaffinity column. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art with respect to any country.

[0003] Aristolochic acids (AAs) are also known as aristolochic acids, oplopanin acids, or aconitine, which are a class of nitrophenyl carboxylic acids. This class of organic compounds naturally exists in plants of Aristolochiaceae, such as Aristolochia and Asarum. Among aristolochic acid compounds, the main toxic components are aristolochic acid I and aristolochic acid II, which are reduced to aristolactam under the catalysis of nitroreductase, and further react with DNA to form an adduct during the reduction process. In the toxicity study of aristolochic acid derivatives on pig kidney tubular epithelial cells, it is found that the toxicity of each derivative is related to its chemical structure. The nitro group is the most important toxic group in aristolochic acid derivatives, and the presence of methoxy and hydroxy groups can further enhance the toxicity of aristolochic acid. Among them, aristolochic acid I is the most toxic component in Aristolochia plants, and traditional Chinese medicine decoction pieces such as Caulis Aristolochiae Mollis, Radix Aristolochiae Debangzhi, and Radix Bupleuri have been prohibited due to the presence of aristolochic acid I. Asarum decoction pieces are strictly limited due to the presence of trace amounts of aristolochic acid I, and traditional Chinese medicine such as Dragon Chine Liver-Draining Pill, Stone-Expelling Granules / Granules, and Coronary Heart Suhe Pill / Capsule are required to modify the prescription, but the mixing of toxic components cannot be completely eliminated.

[0004] Traditional purification methods of aristolochic acid I mostly use organic solvent extraction and chemical adsorption, which have many shortcomings: low specificity, low purification efficiency, poor purification effect; poor experimental sensitivity and repeatability; complicated operation process, long time, high labor intensity; use of organic solvents can cause environmental pollution and harm to operators. The immunoaffinity column method can better solve the above problems, which uses specific immune reaction as the separation means, greatly improves the purity and purification efficiency of the sample, and ensures the reliability and stability of the detection results.

[0005] Immunoaffinity chromatography is a new sample pretreatment technology, which is a combination of immunoresponse and chromatographic analysis method, using the high specificity and affinity of antigen-antibody combination, and using appropriate method to combine specific antigen or antibody to chromatography carrier, so as to effectively separate and purify the respective complementary immune substances. At present, it has been widely used in the analysis of antibodies, hormones, polypeptides, viruses and subcellular compounds. Based on the above research status, the present application considers that for the detection of aristolochic acid I, the immunization affinity method is helpful to realize the enrichment of aristolochic acid I component in the sample, and is of great significance for the safety protection of medicines and decoction pieces. The patent (CN108998424 A) provides a monoclonal antibody of aristolochic acid A, which directly uses aristolochic acid A as a hapten, however, the actual research shows that the direct use of aristolochic acid A as a hapten is insufficient to retain the structure of aristolochic acid I, and the connection arm with the carrier protein is short and the immunogenicity is poor. The patent (CN103387532 A) provides a hapten of aristolochinic lactam, which introduces succinic anhydride to synthesize the hapten succinyl aristolochinic lactam, but the above catalytic reaction needs more than 100 hours, which seriously increases the synthesis cost. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application uses anhydride compounds to modify the structure of aristolochic acid I, first provides a hapten, then connects with the carrier protein to obtain a complete antigen, obtains the corresponding antibody through animal immunization, and further provides an immunoaffinity column, which can realize the efficient enrichment and detection of aristolochic acid I in aristolochia plants and finished drugs.

[0007] Based on the above achievements, the present application provides the following technical scheme: In a first aspect, a hapten of aristolochic acid I is provided, and the structure of the hapten is shown in the following formula (A): (A).

[0008] Aristolochic acid I is a small molecule nitrophenanthrene compound, and its direct use as a hapten is significantly limited in stimulating the immune system. Generally, the effective recognition threshold of the immune system is usually >5000 Da. In the present application, 7-OH-aristolochic acid I is connected with diacid anhydride compounds, which not only maximally retains the characteristic structure of aristolochic acid I, but also introduces carboxyl groups on the original structure, improves the immunogenicity of aristolochic acid I, and is conducive to stimulating the body to complete the immune response, so as to obtain high-quality antibodies. Further, the above hapten is coupled with the carrier protein to form a suitable structure of the connection arm, so as to expose the characteristic structure of aristolochic acid I to the body as much as possible, enhance the immune effect, and improve the affinity of the antibody.

[0009] In a second aspect, the method for preparing the australin hapten of the first aspect comprises the following steps: The 7-OH-austalin I, the catalyst and the dianhydride compound are dissolved in an organic solvent, the reaction is terminated after a period of time under moderate temperature conditions, the product is dissolved by adding water and extracted by a low polarity reagent to obtain the product.

[0010] In the above preparation method, the 7-OH-austalin I, also known as 7-OH-austalin A, has a CAS number of 79185-75-4.

[0011] The catalyst preferably is an acylation catalyst, including but not limited to one or more of 4-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (PPY) and 4-(1-pyrrolidinyl)pyridine. In one embodiment verified by the present application, the catalyst is 4-dimethylaminopyridine (DMAP).

[0012] The organic solvent preferably is a medium polarity or medium strong polarity organic reagent, which should be able to dissolve the reaction product and be miscible with water. In one embodiment verified by the present application, the organic solvent is pyridine. The reaction termination method can be adding a small amount of water, and the volume ratio of water to pyridine is 1:2-4.

[0013] The dianhydride compound includes a chain dianhydride and a cyclic dianhydride. In one embodiment provided by the present application, the dianhydride compound is a cyclic dianhydride, and further is succinic anhydride.

[0014] The moderate temperature condition is preferably a reaction temperature of 45-85°C and a reaction time of 1-6h. Within the parameter range, the lower the temperature, the longer the reaction time is appropriately extended, and the higher the temperature, the longer the reaction time is extended. More desirably, the reaction temperature is 55-75°C and the reaction time is 2-4h.

[0015] Examples of the low polarity reagent include ethyl acetate, trichloromethane or dichloromethane, etc.

[0016] In a third aspect, a complete antigen of austalin I is provided. The complete antigen is a conjugate of the above-mentioned hapten and a carrier protein, and the amino group of the carrier protein is connected to the carboxylic acid group of the hapten by an amide bond.

[0017] Examples of the carrier protein include but are not limited to one of lactoferrin (LF), ovalbumin (OVA), keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).

[0018] In one embodiment of the complete antigen, the carrier protein is bovine serum albumin, and the coupling ratio of the hapten to the bovine serum albumin is 16-20:1. In another embodiment, the carrier protein is ovalbumin, and the coupling ratio of the hapten to the ovalbumin is 7-9:1.

[0019] In a fourth aspect, a method for preparing the complete antigen of aristolochic acid I is provided, and the method comprises the following steps: The hapten of aristolochic acid I, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are added to an organic reagent to obtain a solution A, and a carrier protein is dissolved in a buffer solution to obtain a solution B. The solution A is added to the solution B, and the reaction is carried out at low temperature overnight. The reaction product is dialyzed to obtain the complete antigen.

[0020] In the solution A, the dosage ratio of the hapten, EDC, NHS, and the organic reagent is 4 mg:1-3 mg:1-3 mg:300-500 μL. The organic reagent is exemplified by dimethylformamide (DMF).

[0021] In the solution B, the buffer solution can be PBS, Tris, or the like, and the suitable concentration of the buffer solution is 8-12 mM. The suitable concentration of the carrier protein is 4-6 mg / mL.

[0022] The dosage ratio of the solution A to the solution B is determined according to the coupling ratio of the carrier protein to the hapten or the mass ratio of the hapten to the carrier protein. In a specific embodiment, the carrier protein is bovine serum albumin, and the mass ratio of the hapten to the bovine serum albumin in the solution A and the solution B is 4:8-12. In another embodiment, the carrier protein is ovalbumin, and the mass ratio of the hapten to the bovine serum albumin in the solution A and the solution B is 1:4-6.

[0023] The reaction is carried out at low temperature overnight, and the suitable reaction temperature is 2-5°C.

[0024] In a fifth aspect, a method for preparing an aristolochic acid I antibody is provided, and the method comprises the following steps: using the complete antigen of the fourth aspect to immunize an animal.

[0025] In one embodiment, the aristolochic acid I antibody is a polyclonal antibody, and the preparation method is as follows: using the complete antigen of the fourth aspect to immunize an animal, collecting blood from the animal after the immune titer of the blood of the animal reaches a standard, and separating and purifying the collected blood to obtain the polyclonal antibody.

[0026] In another embodiment, the aristolochic acid I antibody is a monoclonal antibody, and the preparation method is as follows: using the complete antigen of the fourth aspect to immunize an animal, fusing the spleen cells of the immunized animal with myeloma cells, and screening positive clones until a hybridoma cell strain stably secreting the monoclonal antibody is obtained.

[0027] In the above two embodiments, the animal is immunized by injecting the complete antigen of the third aspect mixed with an adjuvant, and the injection is performed once or multiple times.

[0028] The immune titer reaches ≥1:10 5 It is considered to be up to standard.

[0029] In a sixth aspect, an antibody against aristolochic acid I prepared by the method of the fifth aspect is provided; further, the antibody is a polyclonal antibody or a monoclonal antibody.

[0030] In a seventh aspect, a reagent composition is provided, which comprises the hapten of aristolochic acid I of the first aspect, the complete antigen of aristolochic acid I of the third aspect, and / or the antibody of aristolochic acid I of the sixth aspect.

[0031] In an eighth aspect, the hapten of aristolochic acid of the first aspect, the complete antigen of aristolochic acid I of the third aspect, the antibody of aristolochic acid I of the sixth aspect, and the reagent composition of the seventh aspect are used in the detection of aristolochic acid I.

[0032] The above-mentioned use in the detection of aristolochic acid I at least includes the following two aspects: (1) use in the preparation of detection products, including detection kits, detection reagents, and detection instruments, wherein the specific examples of the detection instruments include, but are not limited to, solid-phase extraction columns, extraction fillers, immunosorbent columns, detection test papers, etc. In an embodiment, the polyclonal antibody is used to prepare a solid-phase medium for an immunosorbent column, and the preparation method comprises the following steps: The polyclonal antibody of aristolochic acid I of the sixth aspect is added to the swollen column material, and the reaction is performed at 20-40°C for 0.2-2h with shaking, the column material is then rinsed and ethanolamine is added to block the unbound sites, and then the eluent is used for redissolution.

[0033] In the above-mentioned preparation method, the addition ratio of the polyclonal antibody to the column material is 1-10 mg of antibody per 1 mL of column material.

[0034] In another embodiment, an immunosorbent column is provided, which at least comprises a column body, the column body is integrally formed into a cylindrical shape with a necked end, and in use, the immunosorbent column is placed vertically, has a lower sieve plate close to the variable-diameter portion, and the solid-phase medium is loaded above the lower sieve plate and compacted through an upper sieve plate.

[0035] (2) The application is applied to the detection of aristolochic acid I in a sample to be detected; further, the application provides a pretreatment method for detecting aristolochic acid I in a sample to be detected, which comprises the following steps: adding the sample to be detected into an alcohol solution, uniformly mixing and extracting, reserving the extract, adding aristolochic acid I control sample, uniformly mixing, adding the aristolochic acid I control sample into the above-mentioned immunization affinity column, collecting the eluent, and performing subsequent detection.

[0036] Compared with the prior art, the application has the following beneficial effects: 1. The application provides an artificial antigen of aristolochic acid I, which comprises a hapten and a complete antigen; compared with the artificial antigen of the prior art aristolochic acid derivative, the preparation of the hapten has the advantages of short reaction time, low reaction temperature and economical reaction cost.

[0037] 2. The application provides a corresponding artificial antibody based on the above-mentioned artificial antigen, and further provides an aristolochic acid I immunization affinity column; the aristolochic acid I immunization affinity column can be used for the pretreatment method for detecting aristolochic acid I in aristolochiaceae plants and finished products thereof, reduces the probability of the operator contacting aristolochic acid I, removes most of the interference substances through specific antibody combination with aristolochic acid I, the prepared immunization affinity column has a maximum combination capacity of 2000 ng, and the prepared immunization affinity column can be reused for three times, and the recovery rate is not less than 90%. BRIEF DESCRIPTION OF DRAWINGS

[0038] The drawings accompanying the specification of the application form a part of the application and serve to further understand the application, the illustrative embodiments of the application and the description thereof serve to explain the application and do not constitute an improper limitation of the application.

[0039] Figure 1 The ultraviolet spectrum of the complete antigen described in Examples 2 and 3; Figure 2 The structural schematic diagram of the aristolochic acid I immunization affinity column described in Example 6; 1: upper cover; 2: solid phase extraction empty column; 3: upper sieve plate; 4: solid phase medium; 5: lower sieve plate; 6: lower cover.

[0040] Figure 3 The aristolochic acid I control MRM chromatogram (concentration 105.2 ng / mL) in Examples 7 and 8; Figure 4 The aristolochic acid I MRM chromatogram in the sample of asarum (column addition) in Example 7; Figure 5MRM chromatogram for Aristolochic Acid I in the twenty-five flavor stone pill sample of Example 8; Figure 6 MRM chromatogram for Aristolochic Acid I in the twenty-five flavor stone pill sample (spiked through column) of Example 8; Figure 7 Aristolochic Acid I standard curve graph of Examples 7 and 8; Figure 8 Aristolochic Acid I standard curve graph of Examples 7 and 8; DETAILED DESCRIPTION

[0041] It should be noted that the following detailed description is illustrative only and is not intended to limit the present application in any way. Other embodiments of the present application will readily occur to those skilled in the art. The disclosure of the present application as set forth herein is to be understood against the background of the technical field, wherein all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0043] In the context of the present specification, the word "comprise" is to be interpreted as meaning "comprise, but not limited to." It should not be interpreted as "consist only of."

[0044] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0045] Example 1 In this embodiment, a kind of Aristolochic Acid I hapten and its preparation method are provided, the structure of the Aristolochic Acid I hapten is as shown in the following formula (A): (A).

[0046] The above-mentioned Aristolochic Acid I hapten is synthesized with 7-OH-Aristolochic Acid I and succinic anhydride as raw materials, and the synthesis route is as shown in the following formula: The preparation method steps are as follows: 5 mg of 7-OH-aristolochic acid I, 8 mg of DMAP, and 8 mg of succinic anhydride were weighed and completely dissolved in 300 μL of pyridine. The mixture was magnetically stirred at 65 °C for 3 h, and then 100 μL of ultrapure water was added immediately to terminate the reaction. After drying under nitrogen, 1 mL of ultrapure water was added to dissolve the product, and the mixture was extracted four times with an equal volume of chloroform. After drying under nitrogen, an oily liquid was obtained, which was purified by HPLC to obtain 5.2 mg of aristolochic acid I hapten, with a yield of 81%.

[0047] Example 2 In this embodiment, a complete antigen of aristolochic acid I is provided, which can be used as an immunogen in enzyme immunoassay. It is a complex of aristolochic acid I hapten coupled with a carrier protein, and its structure is shown in formula (B) below. Formula (B).

[0048] The preparation method of the complex shown in formula (B) above is as follows: Take 4 mg of hapten, 2 mg of EDC, and 2 mg of NHS and dissolve them completely in 400 μL of anhydrous DMF to obtain solution A. Stir and react at room temperature for 4 hours. Take 10 mg of bovine serum albumin and dissolve it completely in 2 mL of 10 mM PBS to obtain solution B. Add solution A dropwise to solution B and stir and react overnight at 4°C. After the reaction, dialyze the protein solution with 10 mM PBS for 3 days, changing the medium every 6 hours to obtain aristolochic acid I-bovine serum albumin conjugate, i.e., aristolochic acid I immunogen.

[0049] Example 3 In this embodiment, another complete antigen of aristolochic acid I is provided, which can be used as a coating antigen in enzyme immunoassay. Specifically, it is a conjugate of aristolochic acid I hapten and ovalbumin, and its preparation method is as follows: Take 1 mg of hapten, 1.5 mg of EDC, and 2 mg of NHS and dissolve them completely in 100 μL of anhydrous DMF to obtain solution A. Stir and react at room temperature for 4 hours. Take 5 mg of ovalbumin and dissolve it completely in 1 mL of 10 mM PBS to obtain solution B. Add solution A dropwise to solution B and stir and react overnight at 4°C. After the reaction, dialyze the protein solution with 10 mM PBS for 3 days, changing the medium every 6 hours to obtain aristolochic acid I-ovalbumin conjugate, i.e., aristolochic acid I-coated antigen.

[0050] The coupling results of the complete antigens in Examples 2 and 3 above were identified by ultraviolet spectroscopy, and the results are as follows: Figure 1As shown, the maximum absorption peak of the austrin I hapten-carrier protein is obviously changed compared with the maximum absorption peaks of the austrin I hapten and the carrier protein, indicating that the preparation of the austrin I-carrier protein is successful. The coupling ratio is calculated by using the concentrations of the small molecule and the protein in the conjugate. The coupling ratio of the hapten and BSA is 18:1, and the coupling ratio of the hapten and OVA is 8:1.

[0051] Example 4 In this embodiment, a preparation method of austrin I polyclonal antibody is provided, which comprises the following steps: 1. Animal immunization First immunization: healthy rabbits are selected as immunized animals, the austrin I complete antigen in Example 2 (100 ug per rabbit) is mixed with an equal volume of Freund's complete adjuvant to emulsify to a water-in-oil state (not spread when dropped into water), and is subcutaneously injected at multiple points (neck back, armpit, groin), 50 uL per point, and a total of 6-8 points.

[0052] Boosting immunization: boosting immunization is performed at the 14th, 28th and 42nd days after the first immunization (antigen dose 50 ug per rabbit, and Freund's incomplete adjuvant is used instead. Subcutaneous injection at multiple points (neck back, armpit, groin), 50 uL per point, and a total of 6-8 points.

[0053] 2. Immune titer monitoring Seven days after each boosting immunization, blood is collected from the ear margin vein (non-anticoagulated blood, 1 mL), AA-OVA is coated on a 96-well plate, and the rabbit serum is gradiently diluted (1:10 3 to 1:10 6 ), which is added to the well and incubated for 1 hour. HRP-labeled goat anti-rabbit secondary antibody (1:5000) is added, and the OD450 nm is measured after color development. The titer needs to be ≥1:10 5 to enter the final blood collection.

[0054] 3. Final blood collection and serum separation After the titer reaches the standard, the rabbit is fasted for 12 hours, and blood is collected from the heart or carotid artery (whole blood volume 50-100 mL per rabbit), and the serum is separated.

[0055] 4. Antibody purification The austrin I polyclonal antibody is purified by the method of saturated ammonium sulfate precipitation.

[0056] Example 5 In this embodiment, a preparation method of austrin I monoclonal antibody is provided, which comprises the following steps: 1. Animal immunization Balb / c female mice of 6-8 weeks old are selected, the immunogen is mixed with Freund's adjuvant to emulsify, and the mice are immunized at an immunization dose of 100 ug per mouse to produce antisera.

[0057] 2. Cell fusion and subcloning Take the spleen cells of the successfully immunized Balb / c mice, adjust the ratio of myeloma cells SP2 / 0 and the spleen cells of the immunized mice to 1:5-1:10 for fusion, use the competitive ELISA method to determine the cell culture supernatant, and screen out suitable positive wells. The positive wells are cloned by the limited dilution method until the hybridoma cell strain stably secreting the monoclonal antibody is obtained.

[0058] 3. Preparation and purification of the monoclonal antibody The ascites preparation can also be used, and the steps are as follows: select 10-11 week old Balb / c female mice, inject the adjuvant into the abdominal cavity of the Balb / c mice at 0.3-0.5 ml per mouse, treat the abdominal cavity for about 7-15 days, inoculate the screened hybridoma cell strain into the abdominal cavity of the Balb / c mice after expanding the culture, sacrifice the mice by cervical dislocation, extract the mouse ascites, and centrifuge at 3000 rpm for 10 min.

[0059] Purification of the monoclonal antibody: the ascites is initially purified by the ammonium sulfate precipitation method, and then purified by the Protein G column to obtain the final monoclonal antibody.

[0060] Example 6 In this embodiment, an aristolochic acid I immunoaffinity column is provided, and the structure of the immunoaffinity column is as shown in Figure 2 The immunoaffinity column comprises a column body and a cover body, the column body is integrally formed into a cylindrical shape with a necked end, and in use, the immunoaffinity column is vertically placed, and the lower sieve plate 5 is close to the variable diameter part, the solid phase medium 4 is loaded above the lower sieve plate 5, and is compacted by the upper sieve plate 3. The cover body comprises an upper cover 1 and a lower cover 6, wherein the lower cover 6 covers the necked part, and the upper cover 1 covers the other end of the column body.

[0061] The preparation method of the above-mentioned solid phase medium 4 is as follows: Weigh 1 g of CNBr activated Sepharose-4B column material, swell in 10 mL of 1 mM HCl solution for 30 min, then separate the column material by centrifugation at 5000 rpm for 5 min, wash the swollen matrix with 0.2 M CB pH 9.5 buffer for 3-5 times, 10 mL each time, and separate the column material by centrifugation at 5000 rpm for 5 min each time; ​To the swollen column material, add 40 mg of the polyclonal antibody against austin I prepared in Example 4, 10 mL of 0.2 M CB pH 9.5 buffer, and shake at 37°C for 1 h. Centrifuge the column material, and wash the column material with 0.1 M NaHCO3pH 8.3 buffer for 3-5 times, 10 mL each time. Add 10 mL of 0.1 M ethanolamine containing 0.5 M NaCl pH 8.0 to the column material that has combined the conjugate, and seal the unbound sites at 4°C overnight or at 37°C for 2 h. Wash the column material with eluent 0.1 M sodium acetate, 0.5 M NaCl pH 4.0 and 0.1 M Tris-Cl, 0.5 M NaCl pH 8.0 alternately for 3-5 times, and finally resuspend the column material with 10 mL of eluent 0.1 M Tris-Cl, 0.5 M NaCl pH 8.0 buffer.

[0062] The packing method of the austin I immunoaffinity column is as follows: vertically fix the immunoaffinity column with the constricted neck at the bottom, cover the lower cover 6, put the lower sieve plate 5, and load the above solid phase medium 4 to the upper sieve plate. Gently press the medium until it is not loose, then put the upper sieve plate 3 to compact the medium. Before adding the upper cover, the solid phase extraction column has been added with a column height of 5 / 8-7 / 8 of the storage solution (20% ethanol).

[0063] The maximum column capacity of the austin I immunoaffinity column is determined as follows: use three austin I immunoaffinity columns, and successively add 10 mL of standard solution containing 500 ng / mL austin I to the immunoaffinity columns. Collect the effluent, 1 mL each time, and determine the austin I content by indirect competitive ELISA. It is found that there is only a small amount of austin I in the first 4 tubes, and the austin I content gradually increases from the 5th tube. There is no absorption from the 7th tube. Therefore, the maximum column capacity of the immunoaffinity column is about 4 x 500 ng / mL, i.e. 2000 ng.

[0064] The austin I immunoaffinity column can also be packed with the monoclonal antibody prepared in Example 5, and the packing method is the same as that of the polyclonal antibody.

[0065] In addition, the immunoaffinity column can be regenerated and reused. The regeneration method is as follows: Wash the column tube with PBS (pH 7.4), 1 M NaCl, and 20% ethanol successively, and finally store the column material in 20% ethanol at 4°C. The recovery rate is not less than 90%, and the column material can be reused at least three times.

[0066] Example 7 In this example, a method for detecting austin I in a sample of Asarum based on the austin I immunoaffinity column in Example 6 is provided, which comprises the following steps: 1. Pretreatment of the sample Take 0.05 g of asarum powder in a 50 mL centrifuge tube, add 25 mL of 70% ethanol-water solution, shake and mix, ultrasonic extraction for 30 min. Then centrifuge at 5000 rpm for 10 min. Take 4 mL of supernatant and add 140 uL of 751.2 ng / mL aristolochic acid I control solution and 16 mL of phosphate buffer, shake and mix.

[0067] 2. Sample column operation steps Take out the immunoaffinity column, directly penetrate the lower end of the syringe barrel through the plug above the immunoaffinity column to complete the connection of the syringe barrel and the affinity column, and place it on the pneumatic operation rack for fixation. Take an appropriate amount of pretreated liquid and add it to the syringe. Adjust the pneumatic switch so that the liquid flows out at a rate of 1-2 drops per second. After the sample is added, wash it with 10 mL of eluent (10 mM PBS 1% Tween 20) and then with 10 mL of pure water at a flow rate of 2-3 drops per second. After draining the liquid, elute with 3 mL of eluent (2% acetic acid-methanol) at a flow rate of 1 drop per second, and collect the eluent. After nitrogen blowing, use 0.5 mL of 2% acetic acid-methanol solution to redissolve, pass through a 0.22 um filter membrane, and use it for HPLC or LC-MS instrument analysis (see Figure 4 ).

[0068] Example 8 In this embodiment, a method for detecting aristolochic acid I in twenty-five flavor stone pills based on the aristolochic acid I immunoaffinity column in Example 6 is provided, which includes the following steps: 1. Sample pretreatment Take 1.0 g of sample powder in a 50 mL centrifuge tube, add 25 mL of 70% ethanol-water solution, shake and mix, ultrasonic extraction for 30 min. Then centrifuge at 5000 rpm for 10 min. Take 4 mL of supernatant and add 140 uL of 751.2 ng / mL aristolochic acid I control solution and 16 mL of phosphate buffer, shake and mix.

[0069] 2. Sample column operation steps Take out the immunoaffinity column, directly penetrate the lower end of the syringe barrel through the plug above the immunoaffinity column to complete the connection of the syringe barrel and the affinity column, and place it on the pneumatic operation frame for fixation. Take an appropriate amount of pretreated liquid and add it to the syringe. Adjust the pneumatic switch so that the liquid flows out at a rate of 1-2 drops per second. After the sample is complete, wash 10 mL of the eluent with 10 mL of the eluent (10 mM PBS 1% Tween 20) and 10 mL of pure water at a flow rate of 2-3 drops per second. After draining the liquid, elute with 3 mL of eluent (2% acetic acid-methanol) at a flow rate of 1 drop per second, and collect the eluent. After nitrogen blowing, 0.5 mL of 2% acetic acid-methanol solution is used for redissolution, and after filtration through a 0.22 um filter membrane, it is used for HPLC or LC-MS instrument analysis (see Figure 5 ).

[0070] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A hapten of aristolochic acid I, characterized in that, The hapten structure is shown in the following formula (A): （A）。 2. The method of claim 1, wherein the preparation of the hapten of aristolochic acid I is characterized by, The method comprises the following steps: 7-OH- Aristolochic Acid I, catalyst and succinic anhydride are dissolved in an organic solvent, the reaction is terminated after a period of reaction under a medium temperature condition, the product is dissolved by adding water and extracted by a low-polarity reagent to obtain the product; The catalyst is selected from one or more of 4-dimethylamino pyridine, 4-pyrrolidinyl pyridine and 4- (1-pyrrolidinyl) pyridine; The organic solvent is pyridine; the reaction termination mode is adding water, and the volume ratio of water to pyridine is 1:2-4; The medium temperature condition is 45-85℃, and the reaction time is 1-6h; The low-polarity reagent is selected from ethyl acetate, trichloromethane or dichloromethane.

3. A complete antigen of aristolochic acid I, characterized in that, The complete antigen is a conjugate of the hapten of claim 1 or 2 and a carrier protein, and the two are linked by a covalent bond; the carrier protein is selected from one of lactoferrin, ovalbumin, keyhole limpet hemocyanin or bovine serum albumin.

4. The method of claim 3 for the preparation of the complete antigen of aristolochic acid I, characterized in that, The method comprises the following steps: The hapten of claim 1, EDC and NHS are dissolved in an organic reagent to obtain A liquid, and the carrier protein is dissolved in a buffer solution to obtain B liquid; A liquid is added to B liquid to react overnight under a low temperature, and the reaction product is dialyzed to obtain the product. In A liquid, the dosage ratio of the hapten, EDC, NHS and the organic reagent is 4mg:1-3mg:1-3mg:300-500μL; the organic reagent is dimethylformamide; In B liquid, the buffer solution is selected from PBS or Tris, and the suitable concentration of the buffer solution is 8-12mM; the suitable concentration of the carrier protein is 4-6mg / mL; The reaction temperature under the low temperature is 2-5℃.

5. A method of preparing an antibody to aristolochic acid I, characterized by, The method comprises the following steps: animal immunization is performed by using the complete antigen of claim 3; The antibody is a polyclonal antibody, and the preparation method is as follows: after animal immunization, blood is collected after the immune titer of the blood of the animal reaches a standard, and the collected blood is separated and purified to obtain the antibody; The antibody is a monoclonal antibody, and is prepared by screening positive hybridoma cell secretion or ascites; The animal immunization mode is as follows: the complete antigen of claim 3 is mixed with an adjuvant and then injected, and the injection is performed once or more times; The immune titer reached ≥ 1:10 5 Considered to be met.

6. A reagent composition characterized in that, The composition comprises the hapten of claim 1, the complete antigen of claim 3 or the antibody of claim 5.

7. The hapten of claim 1, the complete antigen of claim 3 or the reagent composition of claim 6 is applied to aristolochic acid I detection.

8. Use according to claim 7, wherein the compound is ###0002### For application in preparation of detection products, the detection products comprise detection kits, detection reagents and detection instruments, wherein the detection instruments are selected from solid-phase extraction columns, extraction fillers, immunization affinity columns and detection test papers.

9. Use according to claim 8, wherein the compound is ###0002### The detection instrument is an immunization affinity column, and the preparation method comprises the following steps: the polyclonal antibody of claim 5 is added to a swollen column material, the column material is shaken and reacted at 20-40℃ for 0.2-2h, the column material is washed and then ethanolamine is added to block unbound sites, and then the column material is redissolved by using an eluent to obtain the product.

10. Use according to claim 9, wherein the compound is ###00010### or a pharmaceutically acceptable salt thereof. The application is used for detecting aristolochic acid I in a sample to be tested; a pretreatment method for the detection includes the following steps: adding the sample to be tested into an alcohol solution, mixing well, extracting and reserving the extract, adding aristolochic acid I control product, mixing, adding into the immunoaffinity column, collecting the eluate, and then detecting.

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