A hapten and artificial antigen for detecting avanafil and their application in the detection of illegal addition in health foods

Abstract

The present invention discloses a hapten for detecting avanafil, an artificial antigen thereof, and their application in the detection of illegal addition in health foods. The antibody prepared in the present invention has a high-sensitivity and high-specificity recognition ability for avanafil. Its half-inhibition concentration for avanafil is 3.34 ng / mL, the quantitative detection range is 1.02 - 10.85 ng / mL, the lowest detection limit is 0.52 ng / mL, and there is no cross-reaction with other structurally and functionally similar substances such as sildenafil and tadalafil. The present invention synthesizes the avanafil hapten, and the method for preparing the avanafil artificial antigen and its antibody is simple and low-cost. The produced avanafil antibody has strong specificity and high sensitivity. At the same time, a more efficient immunoassay method for avanafil is established, which can meet the requirements of on-site detection of a large number of samples and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of detection technologies, and specifically, to a hapten for detecting avanafil, an artificial antigen, and their applications in detecting illegal addition in health foods. Background Art

[0002] Avanafil is a PDE5 inhibitor for treating male erectile dysfunction accidentally invented during the research and development of cardiovascular diseases, and is used for the treatment of male penile erectile dysfunction. The most common adverse reactions of abusing avanafil are headache, flushing, dyspepsia, and muscle pain, followed by nasal congestion, diarrhea, dizziness, and rash. A few cases may have temporary visual color changes (such as inability to distinguish blue and green objects), increased sensitivity of the eyes to light, and other symptoms. It may also cause irreversible damage to the human cardiovascular system, digestive system, respiratory system, urinary system, etc., posing a serious threat to people's health.

[0003] Currently, the determination methods for avanafil in health foods mainly include gas chromatography, gas chromatography - mass spectrometry, liquid chromatography - tandem mass spectrometry, etc. However, although these instrumental methods can effectively analyze avanafil in health foods with high sensitivity and accuracy, they mostly require large - scale instruments, have strong professionalism, and high analysis costs, and cannot meet the needs of on - site rapid detection, restricting their popularization and application in grass - roots detection units. Therefore, establishing a method for rapidly and accurately detecting illegally added avanafil in foods is of great significance for improving the food safety monitoring system.

[0004] The detection principle of immunoassay is to utilize the specific recognition and binding of antigen and antibody, label the antigen or antibody with signal - amplifying molecules such as enzymes, colloidal gold, or fluorescent substances, and use instruments such as enzyme - linked immunosorbent assay (ELISA) readers or fluorescence detectors to qualitatively and quantitatively detect the analyte in the sample. It has greater advantages in on - site detection, with the advantages of high efficiency, rapidity, high sensitivity, strong specificity, convenient detection, low cost, and low skill requirements for operators. However, there is currently no report on the synthetic structure of the avanafil hapten, and there is also a lack of antibodies available for detection. Therefore, it is necessary to establish an efficient avanafil detection method, and it is imperative to search for immune analysis raw materials such as haptens and artificial antigens that can produce antibodies against avanafil. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the above - mentioned defects and deficiencies in the prior art, and to provide a hapten for detecting avanafil, an artificial antigen, and their applications in detecting illegal addition in health foods.

[0006] The first object of the present invention is to provide a hapten for avanafil.

[0007] The second object of the present invention is to provide the use of the above-mentioned avanafil hapten in the preparation of an avanafil artificial antigen.

[0008] The third object of the present invention is to provide an avanafil artificial antigen.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned avanafil artificial antigen in the preparation of an avanafil artificial antibody.

[0010] The fifth object of the present invention is to provide an avanafil artificial antigen combination.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned avanafil artificial antigen combination in the preparation of a reagent for detecting avanafil.

[0012] The seventh object of the present invention is to provide a kit for detecting avanafil.

[0013] The eighth object of the present invention is to provide a method for detecting avanafil.

[0014] In order to achieve the above objects, the present invention is realized by the following solutions:

[0015] The present invention first prepares the hapten AVF-6C of avanafil, and then couples the hapten with lactoferrin (LF), bovine serum albumin (BSA) and chicken ovalbumin (OVA) by the active ester method to obtain the avanafil artificial antigens AVF-6C-LF, AVF-6C-BSA and AVF-6C-OVA. The avanafil artificial antigen AVF-6C-LF is used as an immunogen to immunize animals to obtain avanafil antibodies. At the same time, the avanafil artificial antigens AVF-6C-LF, AVF-6C-BSA and AVF-6C-OVA are used as coating antigens, and the antiserum prepared using AVF-6C-LF as an immunogen is used to select the best combination of immunogen and coating antigen by detecting the titer and inhibition rate of the antiserum.

[0016] Therefore, the present invention claims protection for the following:

[0017] An avanafil hapten AVF-6C, the structural formula of which is shown in formula (I),

[0018]

[0019] The above-mentioned avanafil hapten AVF-6C (the structural formula of which is shown in formula (I)) is named by systematic nomenclature as: (R)-6-(3-chloro-4-methoxybenzyl)(2-(2-hydroxymethyl)pyrrolidin-1-yl)-5-(2-ylmethylcarbamoyl)pyrimidin-4-yl)hexanoic acid.

[0020] The preparation method of the above-mentioned avanafil hapten AVF-6C (the structural formula is shown in formula (I)), and the preparation method includes the following steps:

[0021] S1. React (S)-4-((3-chloro-4-methoxybenzyl)amino)-2-(2-(hydroxymethyl)pyrrolidin-1-yl)-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide and ethyl 6-bromohexanoate fully to obtain product 1;

[0022] S2. Hydrolyze the product 1 obtained in step S1 fully under alkaline conditions, and adjust the pH to acidic to obtain product 2;

[0023] S3. Extract the product 2 obtained in step S2 with ethyl acetate to obtain the hapten AVF-6C of avanafil (the structural formula is shown in formula (I)).

[0024] Preferably, the molar ratio of (S)-4-((3-chloro-4-methoxybenzyl)amino)-2-(2-(hydroxymethyl)pyrrolidin-1-yl)-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide to ethyl 6-bromohexanoate is (1 moL - 1.2 moL):(1.5 moL - 1.8 moL).

[0025] More preferably, the molar ratio of (S)-4-((3-chloro-4-methoxybenzyl)amino)-2-(2-(hydroxymethyl)pyrrolidin-1-yl)-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide to ethyl 6-bromohexanoate is 1 moL:1.5 moL.

[0026] Preferably, step S1 is to fully dissolve 1 moL of (S)-4-((3-chloro-4-methoxybenzyl)amino)-2-(2-(hydroxymethyl)pyrrolidin-1-yl)-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide as a raw material in N,N-dimethylformamide, and then add ethyl 6-bromohexanoate to react fully to obtain product 1;

[0027] The application of the above-mentioned avanafil hapten AVF-6C (the structural formula is shown in formula (I)) in the preparation of avanafil artificial antigen should also be within the protection scope of the present invention.

[0028] An avanafil artificial antigen is obtained by coupling the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with a carrier protein, and its structural formula is shown in formula (II),

[0029]

[0030] wherein, Z is a carrier protein.

[0031] Preferably, the carrier protein is any one of chicken ovalbumin, bovine serum albumin and lactoferrin.

[0032] The use of the above-mentioned artificial avanafil antigen in the preparation of avanafil antibodies should also be within the scope of protection of the present invention.

[0033] The use of the above-mentioned artificial avanafil antigen in detecting avanafil should also be within the scope of protection of the present invention, and the detection is for the purpose of non-disease treatment diagnosis.

[0034] A method for preparing an artificial antigen of avanafil, wherein the hapten of avanafil is coupled to a carrier protein by an active ester method. The carrier protein is coupled to the carboxyl group of the hapten of avanafil (the structural formula of which is shown in formula (I)) by an active ester method.

[0035] Preferably, the active ester method comprises the following steps:

[0036] S1. The avanafil hapten AVF-6C (structural formula as shown in formula (I)), N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-dimethylformamide are fully reacted to obtain solution A; the carrier protein is dissolved in phosphate buffer to obtain solution B;

[0037] S2. After solution A and solution B fully react, they are dialyzed to obtain the artificial antigen for detecting avanafil.

[0038] More preferably, in step S1, the molar ratio of the avanafil hapten AVF-6C (structural formula as shown in formula (I)), N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is (1-1.5):(0.5-1.5):(1-3).

[0039] Further preferably, in step S1, the molar ratio of the avanafil hapten AVF-6C (structural formula as shown in formula (I)), N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1 mol:0.8 mol:1.9 mol.

[0040] More preferably, in step S1, the mass volume ratio of the carrier protein to the phosphate buffer is (10 mg to 20 mg): (2 mL to 4 mL).

[0041] Further preferably, in step S1, the mass volume ratio of the carrier protein to the phosphate buffer is 10 mg:1 mL.

[0042] More preferably, in step S1, the carrier protein is any one of lactoferrin, bovine serum albumin and chicken ovalbumin.

[0043] More preferably, in step S1, avanafil hapten AVF-6C (structural formula shown in formula (I)), N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved in N,N-dimethylformamide, and stirred in the dark at 25 °C for 2-4 h.

[0044] Further preferably, in step S1, 1 mol of avanafil hapten AVF-6C (structural formula shown in formula (I)) is dissolved in 200 μL of N,N-dimethylformamide (DMF) together with 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and stirred in the dark at 25 °C for 4 h.

[0045] More preferably, in step S2, the volume ratio of solution A to solution B is (1-2):(5-10).

[0046] Further preferably, in step S2, the volume ratio of solution A to solution B is 1:5.

[0047] More preferably, in step S2, solution A is added dropwise to solution B, and the reaction is carried out at 3 °C - 5 °C for 10 h - 14 h.

[0048] Further preferably, in step S2, solution A is added dropwise to solution B, and the reaction is carried out at 4 °C for 12 h.

[0049] More preferably, in step S2, the dialysis method is that after the reaction of solution A and solution B is completed, the obtained reaction solution is dialyzed with phosphate buffer solution.

[0050] Further preferably, in step S2, the obtained reaction solution is filled into a dialysis bag and dialyzed with phosphate buffer solution for 2 - 4 days, and the phosphate buffer solution is changed 2 - 4 times a day.

[0051] Most preferably, in step S2, the obtained reaction solution is filled into a dialysis bag and dialyzed with phosphate buffer solution for 3 days, and the phosphate buffer solution is changed 3 times a day.

[0052] The avanafil artificial antigen prepared by the above method should also be within the protection scope of the present invention.

[0053] The application of the above avanafil artificial antigen in detecting avanafil should also be within the protection scope of the present invention, and the detection is for non-disease treatment diagnosis purposes.

[0054] The application of the above avanafil artificial antigen in preparing avanafil antibody should also be within the protection scope of the present invention.

[0055] An avanafil antibody is prepared by immunizing an animal with the above avanafil artificial antigen.

[0056] Preferably, the avanafil antibody is a monoclonal antibody, and the preparation method is to conjugate the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with lactoferrin as an immunogen to immunize an animal to obtain hybridoma cells, culture the obtained hybridoma cells and collect the cells for in vivo proliferation in an animal to obtain ascites, and after identification and purification, the avanafil monoclonal antibody is obtained.

[0057] Preferably, the avanafil antibody is a polyclonal antibody, and the preparation method is to conjugate the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with lactoferrin as an immunogen to immunize an animal, and collect the serum to obtain the polyclonal antibody.

[0058] The application of the above-mentioned avanafil antibody in detecting avanafil should also be within the protection scope of the present invention, and the detection is for non-disease treatment diagnosis purposes.

[0059] The application of the above-mentioned avanafil antibody in preparing a kit for detecting avanafil should also be within the protection scope of the present invention.

[0060] An avanafil artificial antigen combination, comprising an immunogen and a coating antigen, and both the immunogen and the coating antigen are obtained by conjugating the above-mentioned avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with a carrier protein.

[0061] Preferably, the immunogen is obtained by conjugating the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with lactoferrin, and the coating antigen is obtained by conjugating the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with chicken ovalbumin.

[0062] The application of the above-mentioned avanafil artificial antigen combination in detecting avanafil should also be within the protection scope of the present invention, and the detection is for non-disease treatment diagnosis purposes.

[0063] The application of the above-mentioned avanafil artificial antigen combination in preparing a kit for detecting avanafil should also be within the protection scope of the present invention.

[0064] A method for detecting avanafil, using the above-mentioned avanafil artificial antigen combination for detection, and the detection is for non-disease treatment diagnosis purposes.

[0065] A kit for detecting avanafil, comprising an avanafil artificial antigen combination, the avanafil artificial antigen combination comprising an immunogen and a coating antigen, the immunogen being obtained by conjugating the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with lactoferrin, and the coating antigen being obtained by conjugating the avanafil hapten AVF-6C (the structural formula is shown in formula (I)) with chicken ovalbumin.

[0066] Preferably, the kit contains an avanafil antibody, which is obtained by immunizing an animal with lactoferrin conjugated with the avanafil hapten AVF-6C (structural formula shown in formula (I)) as an immunogen.

[0067] More preferably, the antibody is a polyclonal antibody, which is obtained by using lactoferrin conjugated with the avanafil hapten AVF-6C (structural formula shown in formula (I)) as an immunogen to collect serum, and then the polyclonal antibody can be obtained by further purifying the serum using the caprylic acid-ammonium sulfate method.

[0068] Preferably, the kit further contains an enzyme-linked immunosorbent assay (ELISA) plate, a standard of avanafil, and a substrate chromogenic solution.

[0069] More preferably, the ELISA plate is coated with the coating antigen.

[0070] Further preferably, the ELISA plate is coated with a coating antigen obtained by conjugating the avanafil hapten AVF-6C (structural formula shown in formula (I)) with chicken ovalbumin.

[0071] More preferably, the substrate chromogenic solution contains urea peroxide and tetramethylbenzidine.

[0072] More preferably, the kit further contains a termination solution, a washing solution, a blocking solution, an enzyme conjugate concentrate, and an enzyme conjugate diluent.

[0073] Further preferably, the volume fraction of the termination solution is 8% - 12% H2SO4.

[0074] Most preferably, the volume fraction of the termination solution is 10% H2SO4.

[0075] Further preferably, the washing solution is a phosphate buffer solution containing 0.5% - 1.0% (v / v) Tween-20, 0.01% - 0.03% (w / w) sodium azide as a preservative, and 0.1 mol / L - 0.3 mol / L phosphate, with a pH value of 7.2 - 7.6.

[0076] Most preferably, the washing solution is a phosphate buffer solution containing 0.8% (v / v) Tween-20, 0.02% (w / w) sodium azide as a preservative, and 0.2 mol / L phosphate, with a pH value of 7.4.

[0077] Further preferably, the blocking solution is a phosphate buffer solution containing 1% - 3% (w / w) casein and 0.1 mol / L - 0.3 mol / L phosphate, with a pH value of 7.1 - 7.5.

[0078] Most preferably, the blocking solution is a phosphate buffer solution containing 2% (w / w) casein and 0.2 mol / L phosphate, with a pH value of 7.3.

[0079] Further preferably, the enzyme conjugate concentrate is a horseradish peroxidase-labeled goat anti-rabbit antibody or a goat anti-mouse antibody.

[0080] Further preferably, the enzyme conjugate diluent is a phosphate buffer solution with a concentration of 0.1 mol / L to 0.3 mol / L.

[0081] Most preferably, the enzyme conjugate diluent is a 0.2 mol / L phosphate buffer solution.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] The present invention discloses a hapten for detecting avanafil, an artificial antigen thereof, and an application in the detection of illegal addition in health foods. The antibody prepared by the present invention has a high-sensitivity and high-specificity recognition ability for avanafil, with a half-inhibitory concentration of 3.34 ng / mL for avanafil, a quantitative detection range of 1.02 - 10.85 ng / mL, a minimum detection limit of 0.52 ng / mL, and no cross-reaction with other structurally and functionally similar substances such as sildenafil, tadalafil, desmethylsildenafil, hongdenafil, namoxedil, etc. The present invention simplifies the synthesis steps of the avanafil hapten, and the method for preparing the avanafil artificial antigen and its antibody is simple and low-cost. The produced avanafil antibody has strong specificity and high sensitivity. At the same time, a more efficient immunoassay method for avanafil is established, which can meet the requirements of on-site detection of a large number of samples and has broad application prospects. Description of the Drawings

[0084] Figure 1 It is a synthetic route diagram of the avanafil hapten AVF-6C.

[0085] Figure 2 It is an ultraviolet full-wavelength scanning identification result diagram of AVF-6C, AVF-6C-BSA, and BSA.

[0086] Figure 3 It is an ultraviolet full-wavelength scanning identification result diagram of AVF-6C, AVF-6C-OVA, and OVA.

[0087] Figure 4 It is an ultraviolet full-wavelength scanning identification result diagram of AVF-6C, AVF-6C-LF, and LF.

[0088] Figure 5 It is an indirect competitive ELISA standard curve diagram for the antibody used to detect avanafil.

[0089] Figure 6Schematic structural diagram of the avanafil colloidal gold rapid test strip according to Embodiment 9 of the present application, including: PVC plastic bottom plate, sample pad, NC membrane, absorbent pad, test line, and control line.

[0090] Figure 7 Result determination diagram of the avanafil colloidal gold rapid test strip according to Embodiment 9 of the present application; where: A is the detection result of a valid negative sample, B is the detection result of a valid positive sample, and C and D are invalid detection results. Detailed implementation manners

[0091] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0092] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0093] Example 1 Synthesis and identification of avanafil hapten

[0094] I. Experimental method

[0095] The synthesis route of the avanafil hapten AVF-6C is as Figure 1 shown, and the specific steps are as follows:

[0096] 600 mg of (S)-4-((3-chloro-4-methoxybenzyl)amino)-2-(2-hydroxymethyl)pyrrolidin-1-yl)-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide (CAS No.: 330784-47-9) was fully dissolved in N,N-dimethylformamide, and then ethyl 6-bromohexanoate with a molar ratio of 1:2 to the raw material was added, and the whole system was stirred and reacted at room temperature of 25 °C.

[0097] The reaction was detected by TLC, and the developing agent was methanol-ethyl acetate (1:5, v / v). After 12 h of reaction, the reactants were separated and purified by silica gel column chromatography, and gradient elution was carried out with methanol-ethyl acetate (1:5, v / v) as the mobile phase to obtain Product 1. The product was dissolved in an alcohol solvent, water and an aqueous sodium hydroxide solution were added, and the mixture was stirred and hydrolyzed at room temperature. After completion, the pH of the solution was adjusted to acidic to obtain the hapten AVF-6C.

[0098] The hapten AVF-6C was identified by nuclear magnetic resonance and mass spectrometry.

[0099] II. Experimental results

[0100] The nuclear magnetic resonance hydrogen spectrum result of the avanafil hapten AVF-6C was: 11H NMR (400 MHz, dmso) δ 8.70 - 8.66 (t, J = 4.5 Hz, 3H), 7.61 (s, 1H), 7.37 (s, 1H), 7.21 (dd, J = 8.4, 2.0 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 4.72 (s, 2H), 4.48 (dd, J = 30.2, 5.7 Hz, 1H), 4.22 (d, J = 42.0 Hz, 2H), 3.88 (s, 3H), 3.49 (dd, J = 10.8 Hz, 1H), 3.49 (t, J = 6.4 Hz, 2H), 3.21 (dd, J = 12.2 Hz, 1H), 3.19 (dd, J = 4.8 Hz, 1H), 3.11 (m, 1H), 3.05 (dd, J = 4.2 Hz, 1H), 2.23 (t, J = 7.2 Hz, 2H), 1.82 - 1.67 (d, J = 7.7 Hz, 4H), 1.54 - 1.51 (m, 4H), 1.33 (m, 2H).

[0101] The mass spectrometry result of the hapten AVF-6C of avanafil is as follows: MS: C 29 H 36 ClN7O5: 597.2, ESI + [M - H] + : 598.1.

[0102] It can be seen from the mass spectrometry and NMR results that the positive ion molecular peak of (AVF-6C) has a calculated relative molecular mass of 598.2, which is consistent with the actual relative molecular mass, indicating that the hapten AVF-6C of avanafil has been successfully prepared, and its structural formula is as shown in formula (Ⅰ):

[0103]

[0104] The hapten AVF-6C of avanafil is named by systematic nomenclature as: (R)-6-(3-chloro-4-methoxybenzyl)(2-(2-hydroxymethyl)pyrrolidin-1-yl)-5-(2-ylmethylcarbamoyl)pyrimidin-4-yl)hexanoic acid.

[0105] Example 2 Synthesis and Identification of Avanafil Artificial Antigen

[0106] I. Experimental Method

[0107] The hapten AVF-6C of avanafil prepared in Example 1 (structural formula as shown in formula (Ⅰ)) was coupled with lactoferrin (LF), bovine serum albumin (BSA), and chicken ovalbumin (OVA) by the active ester method. The specific steps are as follows:

[0108] Dissolve 1 mol of the avanafil hapten AVF-6C (structural formula shown in formula (I)) with 0.8 mol of N-hydroxysuccinimide (NHS) and 1.9 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 200 μL of N,N-dimethylformamide (DMF), and stir in the dark at room temperature of 25 °C for 4 h to obtain the activated solution of hapten AVF-6C, denoted as solution A; dissolve 10 mg of lactoferrin (LF) in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) completely, denoted as solution B.

[0109] Slowly add 200 μL of solution A drop by drop to 1 mL of solution B, and react at 4 °C for 12 h; put the reaction solution into a dialysis bag and dialyze with PBS buffer for 3 days, 3 times a day. After dialysis, collect the solution in the dialysis bag to obtain the avanafil artificial antigen AVF-6C-LF, aliquot it into centrifuge tubes, and store it at -20 °C for use.

[0110] Among them, the formula of the PBS buffer: 2.90 g of Na2HPO4·12H2O, 8.50 g of NaCl, 0.20 g of KCl, 0.20 g of KH2PO4, and make up to 1000 mL with distilled water.

[0111] The preparation methods of AVF-6C-BSA and AVF-6C-OVA are similar to the synthesis method of the above avanafil artificial antigen AVF-6C-LF, the difference is that BSA or OVA is used instead of LF, and the avanafil hapten AVF-6C (structural formula shown in formula (I)) prepared in Example 1 is used to synthesize the avanafil artificial antigens AVF-6C-BSA and AVF-6C-OVA.

[0112] Identify the avanafil artificial antigens AVF-6C-LF, AVF-6C-BSA and AVF-6C-OVA by the ultraviolet full-wavelength method (150 - 400 nm) respectively.

[0113] 2. Identification of artificial antigen

[0114] Take the above BSA, OVA, LF, AVF-6C (structural formula shown in formula (I)), AVF-6C-LF, AVF-6C-BSA and AVF-6C-OVA, and identify them by the ultraviolet full-wavelength method (150 - 400 nm) respectively.

[0115] The identification results of ultraviolet full-wavelength scanning are as Figures 2 to 4 shown. By comparing the highest absorbance values of LF, BSA and OVA before and after coupling, it can be seen that the absorption curve of AVF-6C-BSA is significantly different from the BSA absorption curve ( Figure 2), the absorption curve of AVF-6C-OVA is significantly different from that of OVA. Figure 3 ), the absorption curve of AVF-6C-LF is significantly different from that of LF. Figure 4 ), the absorption curves of AVF-6C-LF, AVF-6C-BSA, and AVF-6C-OVA show different degrees of deviation from the absorption curves of LF, BSA, and OVA at 280 nm; thus, it can be seen that the avanafil hapten AVF-6C (structural formula as shown in formula (Ⅰ)) is successfully conjugated with LF, BSA, and OVA respectively, and the avanafil artificial antigens AVF-6C-LF, AVF-6C-BSA, and AVF-6C-OVA are successfully prepared in this invention.

[0116] The general structural formula of the avanafil artificial antigen is as shown in formula (Ⅱ),

[0117]

[0118] wherein, Z is the carrier protein OVA, BSA, or LF.

[0119] Example 3 Preparation of Polyclonal Antibodies for Detecting Avanafil

[0120] 1. Animal Immunization

[0121] Mix the avanafil artificial antigen AVF-6C-LF (structural formula as shown in formula (Ⅱ), where Z is lactoferrin) prepared in Example 2 with an immune adjuvant (Freund's complete adjuvant for primary immunization; Freund's incomplete adjuvant for booster immunization) in a volume ratio of 1:1 and emulsify evenly for standby.

[0122] For primary immunization, two healthy 6-week-old New Zealand white rabbits (one male and one female) are injected subcutaneously at multiple points on the back, and the immunization dose for each rabbit is 500 μg of immunoprotein; then, booster immunization is carried out every three weeks for a total of four times.

[0123] One week after the third immunization, blood is taken from the ear vein of the rabbit, centrifuged to obtain the supernatant, and stored at -20 °C for ELISA detection of the immune effect.

[0124] 2. Obtaining Polyclonal Antibodies

[0125] After the fifth immunization, blood is taken from the rabbit's heart. After the obtained rabbit blood is incubated at 37 °C for 2 h, it is placed overnight (12 h) at 4 °C. The next day, the aqueous supernatant is taken out, and then centrifuged at 3000 r / min for 10 min at 4 °C to remove the precipitate, and the supernatant is obtained, which is the polyclonal antibody of avanafil. It is aliquoted, labeled, and stored at -20 °C.

[0126] Example 4 Preparation of Monoclonal Antibodies for Detecting Avanafil

[0127] 1. Animal immunization

[0128] Take the artificial antigen AVF-6C-LF of avanafil prepared in Example 2 (the structural formula is shown in Formula (Ⅱ), where Z is lactoferrin) as the immunoprotein, at 50 μg equivalent / animal, dilute it to 50 μL with PBS, mix it with 50 μL of Freund's adjuvant in a 2.5 mL syringe, and emulsify it with an emulsifier until the emulsion does not spread in the water surface test, and set aside.

[0129] Use Balb / C female mice as immunized animals, and perform multiple-point injection (100 μL / animal) on their abdomen and back according to the immunoadjuvant and immunization cycle shown in Table 1.

[0130] Table 1 Immunization protocol of Balb / C mice with Freund's adjuvant

[0131]

[0132] 2. Detection of animal antibody production

[0133] One week after the third immunization (i.e., the second booster immunization) and each subsequent immunization, collect blood from the tail vein of the mice, centrifuge it to obtain antiserum. Use the icELISA method to check the antibody titer and inhibition rate of the antiserum. The results are shown in Table 2.

[0134] Table 2 Titer and inhibition rate of mouse antiserum

[0135]

[0136] 3. Cell fusion

[0137] (1) Resuscitation and collection of myeloma cells

[0138] Take out the myeloma cells from the -80 °C refrigerator, quickly put them into a 37 °C water bath to thaw, centrifuge the thawed cells at a speed of 1000 r / min for a total of 7 min, and discard the cell cryopreservation solution; use a pipette to aspirate the basal medium to disperse the cells, and continue to centrifuge after dispersion at a speed of 1000 r / min for a total of 7 min, and discard the basal medium; use a pipette to take out and mix it with the complete medium and then put it into a 9 cm culture dish, and expand the culture.

[0139] On the day of cell fusion, remove the supernatant of the myeloma cells in the culture dish, add 25 mL of fresh basal medium using a bent pipette, and blow the adherent myeloma cells repeatedly in a certain direction until the bottom of the culture plate is no longer blurred and becomes transparent. Collect all the medium, seal it and centrifuge it at a speed of 1000 r / min for a total of 8 min. Discard the supernatant after centrifugation to obtain myeloma cells.

[0140] (2) Booster immunization

[0141] Three days before cell fusion, the mice were boost-immunized with an injection concentration of 1 mg / mL and an injection volume of 100 μL without adjuvant.

[0142] (3) Obtaining immune spleen cells

[0143] Add 25 mL of basal medium to a culture dish, place a disposable cell grinding mesh in the culture dish for later use.

[0144] The immunized mice were sacrificed by enucleation of the eyeballs and the blood was collected. After soaking in 75% (v / v) alcohol for 4 min, they were transferred to a laminar flow hood and the spleens were removed.

[0145] Place the removed spleen in the disposable cell grinding mesh in the culture dish. First, use a syringe to draw the medium and inject it into the spleen, repeatedly injecting to blow out the cells until the spleen color changes from red to transparent. Then, gently grind the spleen with the piston of the syringe on the mesh and aspirate the medium to wash the sieve.

[0146] Collect all the medium, seal it and centrifuge at 1000 r / min for 7 min. Discard the supernatant to obtain immune spleen cells.

[0147] (4) Cell fusion

[0148] Mix the myeloma cells and immune spleen cells, add 25 mL of basal medium, seal it and centrifuge at 1000 r / min for 7 min. Discard the supernatant.

[0149] Gently flick the precipitated cells with your finger, place the centrifuge tube in warm water at 37 °C, use a pipette tip to aspirate 1 mL of polyethylene glycol (PEG2000) preheated to 37 °C, and slowly add PEG2000 to the precipitated cells within 1 min. Stir gently with the pipette tip after each drop of PEG2000 is added to mix evenly. Let it stand for 0.5 min. Preheat the basal medium, add 1.5 mL of basal medium within 1 min while gently stirring, add it along the wall, then add basal medium and make up to 20 mL within 5 min with slow up-and-down agitation. Seal the centrifuge tube and centrifuge at 1000 rpm for 7 min, pour out the supernatant, add it to 200 mL of complete medium, gently aspirate and release the liquid with a bent pipette, gently stir the cells, and spread the complete medium containing the fused cells onto a 24-well plate at 2 mL / well and continue to culture in a cell incubator at 37 °C and 5% CO2.

[0150] (5) Screening and identification of hybridoma cells

[0151] Start observing the cell growth and medium status every day from the 7th day of fused cell culture.

[0152] Table 3 Monoclonal cell culture and screening

[0153]

[0154] As shown in Table 3, on the 10th day or when the cells grow too fast, half of the medium was replaced with HAT medium; on the 13th day, the medium was fully replaced with HAT medium; on the 15th day, half of the medium was replaced with HT medium, and the cell supernatant was tested for the first icELISA test to determine whether the cells were fused and the degree of cell fusion; on the 17th day, the HT medium was fully replaced, and the second icELISA test was performed; on the 19th day, the HT medium was fully replaced, and the third icELISA test was performed. The results of the previous three supernatant tests were compared, and the high titer wells were selected for limiting dilution. When the icELISA test was performed on the limited dilution cell supernatant, the titer and inhibition rate were used as the measurement indicators. After the positive wells were selected, the next round of limiting dilution was performed until each well on each plate was positive and the titer and inhibition were similar after detection. At this point, the hybridoma cell line was successfully established and frozen in time.

[0155] Example 5 Combination selection of avanafil immunogen and coating agent

[0156] The polyclonal antibody prepared in Example 3 and the avanafil artificial antigen prepared in Example 2 were used as coating agents, and the best combination of immunogen and coating agent was selected by the serum titer and inhibition rate obtained by the indirect competitive ELISA method. The specific operation steps are as follows:

[0157] 1. The artificial antigens of avanafil, AVF-6C-BSA (structural formula as shown in formula (II), wherein Z is bovine serum albumin), AVF-6C-OVA (structural formula as shown in formula (II), wherein Z is chicken ovalbumin) and AVF-6C-LF (structural formula as shown in formula (II), wherein Z is lactoferrin) were diluted to a concentration of 250 ng / mL with coating solution (0.05 M carbonate buffer solution, pH 9.6), 100 μL / well was coated on a 96-well ELISA plate, incubated in a constant temperature water bath at 37°C overnight, the coating solution was discarded, and the plate was washed twice with PBST (0.01 M PBS, 0.06% Tween-20 (v / v));

[0158] 2. Add 120 μL of blocking solution (pH 7.3, containing 2% casein and 0.2 mol / L phosphate buffer) to each well, block at 37°C for 3 h, discard the blocking solution, clap the plate, and dry it in a drying oven at 37°C for later use;

[0159] 3. Dilute the polyclonal antibody prepared in Example 3 to 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000 with PBST, and set up blank control wells (replaced with PBST); dilute the 1 mg / mL avanafil standard product 1000 times with PBST to a concentration of 1 μg / mL;

[0160] 4. For the titer column, first add 50 μL of PBST to each well, then add the polyclonal antibody at different dilution ratios to the wells in sequence at 50 μL per well. Do not add antibody to the last well and replace it with 50 μL of PBST;

[0161] 5. For the inhibition column, first add 50 μL of the avanafil standard product to each well, then add the polyclonal antibody at different dilution ratios to the wells in sequence at 50 μL per well. Do not add antibody to the last well and replace it with 50 μL of PBST;

[0162] 6. Incubate at 37 °C for 40 min, wash 5 times, and tap the plate;

[0163] 7. Add goat anti-rabbit secondary antibody-HRP (diluted 5000 times), incubate at 37 °C for 30 min, wash 5 times, and tap the plate;

[0164] 8. Add the chromogenic solution and incubate at 37 °C for color development for 10 min;

[0165] 9. Add 10% (v / v) H2SO4 to terminate the reaction and read the OD value at 450 nm; count the titer and inhibition rate. The titer is the antibody dilution multiple corresponding to an OD of about 1.0, and the inhibition rate = (OD value of the titer - OD value of the inhibition) / OD value of the inhibition × 100%. 450 The combination results of different immunogens and coating antigens are shown in Table 4.

[0166] The screening results of immunogens and coating antigens are shown in Table 4.

[0167] Table 4 Screening results of immunogens and coating antigens

[0168]

[0169] As can be seen from Table 4, the antiserum produced by immunizing New Zealand white rabbits with the artificial antigen AVF-6C-LF of avanafil (the structural formula is shown in Formula (II), where Z is lactoferrin) as the immunogen all has a certain titer, and the obtained antiserum has different degrees of inhibitory effects on the target analyte avanafil. Among them, the antiserum titer of 1:128000 and the inhibition rate of 87.12% shown by the immunogen and coating antigen structure combination numbered 2 are the best combinations; under this combination, the polyclonal antibody of avanafil can not only recognize the target analyte avanafil, but also has good antibody sensitivity; the antiserum titer and inhibition rate are higher than those of the immunogen and coating antigen combinations numbered 1 and 3, so the immunogen and coating antigen structure combination numbered 2 is the best combination. That is, AVF-6C-LF (the structural formula is shown in Formula (II), where Z is lactoferrin) is used as the immunogen, and AVF-6C-OVA (the structural formula is shown in Formula (II), where Z is ovalbumin) is used as the coating antigen.

[0170] Example 6 Establishment of an indirect competitive ELISA detection method for avanafil

[0171] I. Experimental method

[0172] 1. An indirect competitive ELISA method for detecting avanafil, comprising the following steps:

[0173] (1) Using the artificial antigen AVF-6C-OVA of avanafil prepared in Example 2 (the structural formula is shown in Formula (II), where Z is ovalbumin) as the coating antigen, diluting it to 1 μg / mL with a coating solution (0.05 M carbonate buffer solution, pH 9.6), coating a 96-well enzyme-linked immunosorbent assay (ELISA) plate at 100 μL / well, and incubating overnight (12 h) at 37 °C;

[0174] (2) Discarding the coating solution, washing 2 times, and patting dry;

[0175] (3) Adding 120 μL of a blocking solution (pH value is 7.3, containing 2% casein by mass fraction, 0.2 mol / L phosphate buffer solution) to each well, and blocking at 37 °C for 3 h;

[0176] (4) Discarding the blocking solution, patting the plate, taking it out after drying at 37 °C for 30 min, and packing it in a self-sealing bag for standby;

[0177] (5) Diluting the polyclonal antibody prepared in Example 3 with PBST at 1:16000 times, and diluting the avanafil standard product to 10000 ng / mL, 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, 0.001 ng / mL;

[0178] (6) Add 50 μL of the diluted solution of avanafil (in quadruplicate) to each row, then add 50 μL / well of the antibody dilution solution, incubate at 37 °C for 40 min, and wash 5 times;

[0179] (7) Add goat anti-rabbit secondary antibody-HRP (diluted 5000-fold), incubate at 37 °C for 30 min, wash 5 times, and pat dry;

[0180] (8) Add the chromogenic solution, 100 μL per well, and develop color for 10 min;

[0181] (9) Add 50 μL of 10% (v / v) H2SO4 to terminate the reaction, and read the OD value at 450 nm;

[0182] (10) Draw the ELISA standard curve based on the OD values:

[0183] Take B / B0 as the ordinate (B is the absorbance OD of the standard product of avanafil at different concentrations 450 , B0 is the absorbance OD of the blank control well 450 ), take the logarithm of the standard product concentration as the abscissa, perform curve fitting using the Logistic function to obtain the formula of the standard curve, and prepare the standard curve, as Figure 5 shown.

[0184] II. Experimental Results

[0185] It can be seen from Figure 5 that the avanafil antibody has a high sensitivity and high specificity for recognizing avanafil. Its half-inhibitory concentration for avanafil is 3.34 ng / mL, the quantitative detection range is 1.02 - 10.85 ng / mL, and the lowest detection limit is 0.52 ng / mL.

[0186] Example 7 Specificity Evaluation of Avanafil Antibody

[0187] 1. Test Method

[0188] Determine the specificity of the avanafil antibody through cross-reaction experiments with avanafil and its analogs sildenafil, tadalafil, desmethylsildenafil, hongdenafil, and namoxysildenafil. The specificity of the antibody is expressed by the cross-reaction rate (CR). The smaller the cross-reaction rate, the stronger the specificity. Dilute avanafil and its analogs (sildenafil, tadalafil, desmethylsildenafil, hongdenafil, namoxysildenafil) respectively in a serial dilution, and use the indirect competitive ELISA method for determination. The steps are the same as in Example 6 to obtain the IC 50 values of various analogs. Calculate the avanafil cross-reaction rate (CR) according to the following formula:

[0189] CR(%) = IC 50 (avanafil) / IC50 (Analog) × 100%.

[0190] 2. Experimental results

[0191] The cross - reaction results of avanafil and its analogs are shown in Table 5. It can be seen that the cross - reaction rate of the antibody against avanafil to avanafil is 100%, and the IC 50 is 3.34 ng / mL, and the cross - reaction rates to sildenafil, tadalafil, desmethylsildenafil, hongdenafil, and namoxedil are all < 0.01%; it shows that the antibody used for detecting avanafil has strong specificity, can effectively exclude the interference of its analogs on the detection of avanafil, and can be specifically used for the detection of avanafil.

[0192] Table 5 Cross - reaction results of avanafil and its analogs

[0193]

[0194] Example 8 An ELISA kit for detecting avanafil

[0195] 1. Composition

[0196] (1) Microtiter plate coated with coating antigen:

[0197] The microtiter plate is prepared by the following method:

[0198] Use the avanafil artificial antigen AVF - 6C - OVA (the structural formula is shown in formula (Ⅱ), where Z is chicken ovalbumin) prepared in Example 2 as the coating antigen, dilute it to 125 ng / mL with coating buffer (0.05 M carbonate buffer solution, pH 9.6), add 100 μL per well to the microtiter plate, incubate overnight at 37°C in the dark, remove the liquid in the wells, wash 2 times with the washing solution in this kit, 30 s each time, pat dry, then add 120 μL of blocking solution (pH value is 7.3, containing 2% casein, 0.2 mol / L phosphate buffer) to each well, incubate at 37°C in the dark for 3 h, pour out the liquid in the wells and pat dry, and store it in vacuum - sealed with aluminum film after drying;

[0199] (2) Standard products:

[0200] Standard solutions of 11 different concentrations of avanafil, with concentrations of 10000 ng / mL, 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, 0.128 ng / mL, 0.0256 ng / mL, 0.00512 ng / mL, 0.00102 ng / mL respectively;

[0201] (3) Antibody:

[0202] Polyclonal antibody prepared using the artificial antigen AVF-6C-LF of avanafil (structural formula shown in formula (II), where Z is lactoferrin) as the immunogen, and monoclonal antibody prepared using the artificial antigen AVF-6C-LF of avanafil (structural formula shown in formula (II), where Z is lactoferrin) as the immunogen in Example 4;

[0203] (4) Enzyme conjugate concentrate:

[0204] Goat anti-rabbit antibody or goat anti-mouse antibody labeled with horseradish peroxidase;

[0205] (5) Substrate chromogenic solution:

[0206] Composed of solution A and solution B. Solution A is urea peroxide, and solution B is tetramethylbenzidine;

[0207] (6) Stop solution

[0208] H2SO4 with a volume fraction of 10%;

[0209] (7) Washing solution

[0210] With a pH value of 7.4, containing phosphate buffer solution with a volume fraction of Tween-20, a mass fraction of 0.02% sodium azide preservative, and 0.2 mol / L;

[0211] (8) Enzyme conjugate diluent

[0212] 0.2 mol / L phosphate buffer solution.

[0213] 2. Usage

[0214] (1) Sample detection

[0215] Number the corresponding micro wells of the samples and the standards of this kit in sequence. Make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Dilute the enzyme conjugate concentrate with the enzyme conjugate diluent at a volume ratio of 1:10 as needed (that is, add 1 part of the enzyme conjugate concentrate to 10 parts of the enzyme conjugate diluent, and prepare it for use immediately) to obtain the enzyme conjugate working solution.

[0216] Add 50 μL of the standard or sample to the corresponding micro well, then add 50 μL of the enzyme conjugate working solution, gently shake and mix well, cover with a cover film, and react in a dark environment at 25 °C for 30 min.

[0217] Discard the liquid in the well by centrifugation, and add 250 μL / well of the washing working solution. Wash thoroughly 4 - 5 times, with an interval of 10 s each time. Discard the washing solution in the plate wells, and pat dry with absorbent paper (the bubbles not removed after patting dry can be pricked with an unused pipette tip).

[0218] Add 50 μL / well of Substrate Chromogenic Solution A, then add 50 μL / well of Substrate Chromogenic Solution B. Gently mix by oscillation. Cover with a cover film and react in a dark environment at 25 °C for 10 min. Add 50 μL / well of Stop Solution, gently mix by oscillation. Set the microplate reader at 450 nm and measure the OD value of each well.

[0219] (2) Plotting the standard curve

[0220] Plot the ELISA standard curve based on the OD values of the avanafil standard at 450 nm. The specific method is as follows:

[0221] Use B / B0 as the ordinate (B is the absorbance OD of avanafil standards at different concentrations 450 , and B0 is the absorbance OD of the blank control well 450 ), and the logarithm of the standard concentration as the abscissa. Use the Logistic function for curve fitting to obtain the formula of the standard curve and prepare the standard curve.

[0222] (3) Calculation of sample concentration

[0223] Substitute the average value of the sample OD 450 into the formula of the above standard curve to obtain the concentration of the sample, and then multiply by its corresponding dilution factor to obtain the actual concentration of avanafil in the test sample.

[0224] Example 9 A colloidal gold test strip for detecting avanafil

[0225] 1. Preparation of gold-labeled antibody and gold-labeled conjugate pad

[0226] Prepare a colloidal gold suspension with an average diameter of 40 nm by the method of reducing chloroauric acid with trisodium citrate.

[0227] First, adjust the pH of the colloidal gold to 8.5 with 0.2 mol of K2CO3 solution, then use the classical NaCl titration method to determine the antibody labeling amount. Finally, select 20 μg of the antibody of avanafil prepared in Example 3 and 1 mL of colloidal gold solution for labeling to obtain the gold-labeled antibody, and store it at 4 °C.

[0228] As Figure 6 shown, the colloidal gold rapid test strip is composed of an NC membrane (nitrocellulose membrane), a sample pad, a water absorption pad, and a PVC plastic bottom plate stacked together.

[0229] Use a XYZ-3000 three-dimensional spraying film instrument to spray a 4% BSA solution on the glass wool at a rate of 8 μL / cm, dry it in an oven at 42 °C for 50 min, then spray the gold-labeled antibody on the glass wool at a rate of 6 μL / cm, and after drying it in an oven at 42 °C for 50 min, the gold-labeled conjugate pad is obtained and stored in vacuum drying.

[0230] 2. Cellulose membrane coated with conjugated antigen goat anti - rabbit

[0231] Using an XYZ - 3000 three - dimensional film spraying instrument, the coated antigen with a concentration of 1 mg / mL (AVF - 6C - OVA (the structural formula is shown in formula (Ⅱ), where Z is ovalbumin)) is sprayed on the right side of the cellulose membrane at a rate of 1.2 μL / cm in a direction perpendicular to the long side of the cellulose membrane as the test line (i.e., the T line). Using an XYZ - 3000 three - dimensional film spraying instrument, goat anti - rabbit IgG with a concentration of 120 μg / L is sprayed on the left side of the cellulose membrane at a rate of 1.2 μL / cm in a direction perpendicular to the long side of the cellulose membrane as the control line (i.e., the C line). The two lines are spaced 8 mm apart, and thus the cellulose membrane coated with conjugated antigen goat anti - rabbit is obtained.

[0232] 3. Assembly of the rapid test strip

[0233] The cellulose membrane is pasted on the middle part of the backing plate 1; the absorbent pad is pasted on the left side of the cellulose membrane with an overlap of 1 mm; the gold - labeled conjugate pad is pasted on the right side of the cellulose membrane with an overlap of 1 mm; the sample pad is pasted on the right side of the gold - labeled conjugate pad with an overlap of 2 mm, and thus the colloidal gold test strip is obtained. The colloidal gold test strip is cut into strips 3.05 mm wide using a cutting machine.

[0234] 4. Preparation of the test solution

[0235] 0.5 mL of the test sample is aspirated and mixed thoroughly with 9.5 mL of 0.2 mol / L phosphate buffer solution at pH 7.4, and vortex - mixed for 30 s to obtain the test solution.

[0236] 5. Detection and judgment of the rapid test strip

[0237] The test solution is dropped onto the sample pad and left to stand. The test solution drives the test substance and the gold - labeled antibody in the gold - labeled conjugate pad 3 to diffuse towards the cellulose membrane through capillary action and finally penetrate into the absorbent pad.

[0238] Result judgment: The gold - labeled antibody binds to the invisible quality control line (i.e., the C line) on the cellulose membrane, making the quality control line (i.e., the C line) appear red, indicating that the test result is valid. If the quality control line (i.e., the C line) does not appear red, it indicates that the test result is invalid; as Figure 7 shown, if there is avanafil in the sample, avanafil binds to the gold - labeled antibody, thereby occupying the antigen - binding site on the gold - labeled antibody and preventing the gold - labeled antibody from binding to the invisible test line (the conjugate of hapten and carrier protein) on the cellulose membrane, making the invisible test line (i.e., the T line) not appear red or appear very faintly, indicating that the test sample is positive or weakly positive; if there is no avanafil in the sample, when the gold - labeled antibody moves upward, it encounters the invisible test line (i.e., the T line) and shows a clear red line, indicating that the test sample is negative.

[0239] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. An avanafil hapten, characterized in that, Its structural formula is shown in formula (I).

2. Use of the avanafil hapten according to claim 1 in the preparation of an avanafil artificial antigen.

3. An avanafil artificial antigen, characterized in that, It is obtained by conjugating the avanafil hapten according to claim 1 with a carrier protein, and its structural formula is shown in formula (II). Wherein Z is a carrier protein.

4. Use of the avanafil artificial antigen according to claim 3 in the preparation of an avanafil artificial antibody.

5. An avanafil artificial antigen combination, characterized in that, It comprises an immunogen and a coating antigen, and both the immunogen and the coating antigen are obtained by conjugating the avanafil hapten according to claim 1 with a carrier protein.

6. The avanafil artificial antigen combination according to claim 5, characterized in that, The immunogen is obtained by conjugating the avanafil hapten according to claim 1 with lactoferrin, and the coating antigen is obtained by conjugating the avanafil hapten according to claim 1 with chicken ovalbumin.

7. Use of the avanafil artificial antigen combination according to any one of claims 5 to 6 in the preparation of a reagent and / or kit for detecting avanafil.

8. A kit for detecting avanafil, characterized in that, The kit comprises the avanafil artificial antigen combination according to any one of claims 5 to 6.

9. The kit according to claim 8, wherein The kit further comprises an enzyme-labeled plate, a standard product of avanafil and a substrate chromogenic solution.

10. A method for detecting avanafil, characterized in that, Detection is carried out using the artificial antigen combination according to any one of claims 5 to 6, and the detection is for non-disease treatment diagnosis purposes.

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