Lorcaserin artificial hapten, artificial antigen and preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a lorcaserin artificial hapten, an artificial antigen and a preparation method and application of the lorcaserin artificial hapten and the artificial antigen. According to the invention, the lorcaserin hydrochloride is used as a starting material for synthesizing the artificial hapten, and the lorcaserin artificial hapten maximally retains the characteristic structure of lorcaserin, has an active group capable of being coupled with carrier protein, and can be used as an antigenic determinant; the further prepared lorcaserin artificial antigen can be immunized to obtain an anti-lorcaserin artificial antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit reaches up to 1: 256000, and the lorcaserin artificial antigen can be used for rapid and accurate immunodetection and immunoassay of lorcaserin.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to a lorcaserin artificial hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Lorcaserin is a novel weight loss drug that acts on the central nervous system. It selectively stimulates the hypothalamic satiety center to reduce food intake and promote satiety. Therefore, it is generally used to treat binge eating and obesity. Because of its more and more significant side effects, ordinary people who want to lose weight should not use it at will. If you need to use these drugs, you must do so under the guidance of a doctor and avoid buying them on your own to avoid serious consequences. Its adverse reactions mainly include headache, dizziness, fatigue, nausea, dry mouth, constipation, etc.

[0003] Lorcaserin has a certain impact on the cardiovascular system. Therefore, it has a risk of cancer for patients who already have cardiovascular diseases, such as heart valve disease, arrhythmia, etc., as well as patients at risk of cardiovascular diseases. Excessive abuse will lead to the following pains: kidney necrosis, hemodialysis, abnormal liver function, wearing diapers all day, etc.

[0004] At present, most of the methods used in China for analysis and determination are high performance liquid chromatography, gas chromatography, mass spectrometry, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, infrared spectrum, etc., which are not suitable for large-scale sampling and analysis due to the high cost of use and maintenance. Immunoassay using artificial synthetic antigens has the advantages of convenient operation, strong specificity, and fast determination speed. Therefore, it is necessary to synthesize an artificial hapten and artificial antigen of lorcaserin to meet the needs of immunoassay. At present, there is still no relevant report on artificial hapten and artificial antigen of lorcaserin.

[0005] Therefore, it is necessary to develop an economical, simple and rapid detection method to detect the level of lorcaserin in the human body, and the preparation of artificial lorcaserin antigen is the basis for the realization of this method. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a lorcaserin artificial hapten, an artificial antigen and a preparation method thereof, and applies the lorcaserin artificial antigen to the preparation of an anti-lorcaserin antibody with high detection sensitivity and strong specificity.

[0007] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A lorcaserin artificial hapten, whose molecular structure is shown in formula (I): Formula (I).

[0008] The method for preparing the above-mentioned artificial hapten of lorcaserin comprises the following steps: (S.1) dissolving lorcaserin hydrochloride in deionized water, and adjusting the pH to alkaline with aqueous ammonia to obtain an alkaline solution; (S.2) extracting the alkaline solution obtained in step (S.1) with dichloromethane, collecting the dichloromethane phase, drying, filtering, and drying to obtain a colorless oil A; (S.3) dissolving the colorless oil A obtained in step (S.2) in a solvent, adding potassium carbonate, and then adding ethyl bromoacetate, mixing evenly and heating and stirring to react, filtering after the reaction is complete, drying to obtain a colorless oil B; (S.4) Prepare sodium hydroxide solution and hydrochloric acid solution respectively, then add tetrahydrofuran, methanol and the prepared sodium hydroxide solution to the colorless oil B obtained in step (S.3), heat and stir to react until the reaction is complete, adjust the pH to acidic with the prepared hydrochloric acid solution, dry it, extract with anhydrous ethanol, filter, dry it, separate, and obtain lorcaserin artificial hapten I.

[0009] The present invention uses lorcaserin hydrochloride as the starting material for synthesizing the artificial hapten of lorcaserin, which not only retains the characteristic structure of lorcaserin to the greatest extent, but also has an active group that can be coupled with a carrier protein and can be used as an antigenic determinant. The present invention introduces a connecting arm at the imino position of lorcaserin, and the introduction of the connecting arm at the modification site can retain the characteristic structure of lorcaserin to the greatest extent. Compared with the use of a cyclic connecting arm, the present invention uses a straight chain as the connecting arm, which can reduce non-specific binding during immunoassay and can also reduce the immune response to the connecting arm generated during immunization, thereby increasing the probability of generating specific antibodies.

[0010] Preferably, the pH of the alkaline solution in step (S.1) is 8-10.

[0011] As further preferred, the pH of the alkaline solution in step (S.1) is 9.

[0012] Preferably, the molar ratio of the colorless oil A added in step (S.3) to potassium carbonate is 1:(3.5-4.5).

[0013] As further preferred, the molar ratio of the colorless oil A added in step (S.3) to potassium carbonate is 1:4.

[0014] Preferably, the molar ratio of the colorless oil A added in step (S.3) to ethyl bromoacetate is 1:(1.5-2.5).

[0015] As further preferred, the molar ratio of the colorless oil A added in step (S.3) to ethyl bromoacetate is 1:2.

[0016] Preferably, the solvent in step (S.3) is any one of acetone, N,N-dimethylformamide and anhydrous ethanol.

[0017] Preferably, the solid-liquid ratio of the colorless oil A added in step (S.3) to the solvent is 9-11:0.5-1.5 mg / mL.

[0018] As a further preference, the solid-liquid ratio of the colorless oil A added in step (S.3) to the solvent is 10:1 mg / mL.

[0019] Preferably, the solid-liquid ratio of the colorless oil B, tetrahydrofuran, methanol and sodium hydroxide solution added in step (S.4) is 90-110:1.5-3:2-3:8-12 mg / mL / mL / mL.

[0020] As further preferred, the solid-liquid ratio of the colorless oil B, tetrahydrofuran, methanol, and sodium hydroxide solution added in step (S.4) is 100:2.02:2.31:10 mg / mL / mL / mL.

[0021] Preferably, the concentration of the sodium hydroxide solution prepared in step (S.4) is 0.5-2 mol / L; the concentration of the hydrochloric acid solution prepared is 0.5-1.5 mol / L.

[0022] As further preferred, the concentration of the sodium hydroxide solution prepared in step (S.4) is 1 mol / L; the concentration of the hydrochloric acid solution prepared is 1 mol / L.

[0023] Preferably, in step (S.4), the pH value is adjusted to 2-4 using a prepared hydrochloric acid solution.

[0024] As a further preference, in step (S.4), the pH is adjusted to 3 using the prepared hydrochloric acid solution.

[0025] A lorcaserin artificial antigen, whose molecular structure is shown in formula (II): Wherein, BSA is bovine serum albumin, and n is 55 to 60. Preferably, n is 57.

[0026] The method for preparing the above-mentioned lorcaserin artificial antigen comprises the following steps: The artificial hapten I of lorcaserin as described above was coupled with bovine serum albumin by using the N-hydroxysuccinimide active ester method, and the artificial hapten II of lorcaserin was obtained after purification.

[0027] Preferably, the method for preparing the above-mentioned artificial lorcaserin antigen comprises the following steps: (a) Mixing lorcaserin artificial hapten I, N-hydroxysuccinimide and cyclohexylcarbodiimide in N,N-dimethylformamide, heating and stirring until the reaction is complete, centrifuging and collecting the supernatant; (b) adding the supernatant obtained in step (a) to the bovine serum albumin solution, mixing evenly and standing overnight, dialyzing and centrifuging to obtain the supernatant to obtain lorcaserin artificial antigen II.

[0028] Preferably, the molar ratio of the lorcaserin artificial hapten, N-hydroxysuccinimide and cyclohexylcarbodiimide added in step (a) is 1:(1.35-1.5):(1.35-1.5).

[0029] As a further preference, the molar ratio of the lorcaserin artificial hapten, N-hydroxysuccinimide and cyclohexylcarbodiimide added in step (a) is 1:1.35:1.35.

[0030] Preferably, the solid-liquid ratio of the lorcaserin artificial hapten to N,N-dimethylformamide added in step (a) is 15-25:0.5-1.5 mg / mL.

[0031] As a further preference, the solid-liquid ratio of the lorcaserin artificial hapten to N,N-dimethylformamide added in step (a) is 20:1 mg / mL.

[0032] Preferably, the reaction temperature of the heating and stirring reaction process in step (a) is 20 to 30° C., and the reaction time is 18 to 20 hours.

[0033] As further preferred, the reaction temperature of the heating and stirring reaction process in step (a) is 25° C. and the reaction time is 18 h.

[0034] Preferably, the volume ratio of the supernatant added in step (b) to the bovine serum albumin solution is 0.9-1.1:9-11.

[0035] As further preferred, the volume ratio of the supernatant added in step (b) to the bovine serum albumin solution is 1:10.

[0036] Preferably, the concentration of the bovine serum albumin solution added in step (b) is 4-6 mg / mL.

[0037] As further preferred, the concentration of the bovine serum albumin solution added in step (b) is 5 mg / mL.

[0038] As further preferred, the method for preparing the bovine serum albumin solution comprises the following steps: Bovine serum albumin is dissolved in 0.01 M PBS buffer, wherein the pH of the PBS buffer is 7.2-7.4.

[0039] The present invention uses bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared with bovine gamma globulin (BGG): ① Bovine serum albumin can better bind to the lorcaserin artificial hapten, thereby preparing lorcaserin artificial antigens with different coupling ratios; ②From the experiment, the stability of the artificial hapten of lorcaserin is stronger after it is combined with bovine serum albumin. After seven days of stability evaluation, it can be known that: at the same protein concentration, the combination of bovine serum albumin and the artificial hapten of lorcaserin does not cause protein precipitation, which is more stable; while the combination of bovine immunoglobulin and the artificial hapten of lorcaserin produces a large amount of protein precipitation after stability evaluation, so the combination of bovine immunoglobulin and the artificial hapten of lorcaserin is easy to separate in the subsequent treatment, so that the artificial antigen prepared in the end is easy to precipitate during long-term storage, and the stability is poor. The combination of bovine serum albumin and the artificial hapten of lorcaserin can be stored for a long time at different pH and temperature, and the protein itself is not easy to separate, which is more stable; ③ Compared with bovine immunoglobulin G, the specificity of bovine serum albumin combined with lorcaserin artificial hapten is stronger; ④ Compared with bovine immunoglobulin G, the bovine serum albumin used in the present invention is cheap and easy to obtain, and can reduce costs to a certain extent.

[0040] Preferably, the temperature for standing overnight in step (b) is 3-5°C.

[0041] As further preferred, the temperature for standing overnight in step (b) is 4°C.

[0042] Use of the above-mentioned artificial lorcaserin antigen in preparing anti-lorcaserin antibodies.

[0043] An anti-lorcaserin artificial antibody is obtained by immunizing an animal with the above-mentioned lorcaserin artificial antigen and can generate a specific immune reaction with lorcaserin.

[0044] The present invention has been found through repeated experiments that the titer of the immune serum obtained by immunizing New Zealand white rabbits with the artificial lorcaserin antigen is 1:256000. This shows that the artificial lorcaserin antigen of the present invention can be used to immunize and obtain anti-lorcaserin antibodies with high affinity, high sensitivity and strong specificity, and the anti-lorcaserin antibodies can be used for immunodetection and analysis of lorcaserin.

[0045] Therefore, the present invention has the following beneficial effects: The artificial lorcaserin hapten of the invention retains the characteristic structure of lorcaserin to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant cluster; the artificial lorcaserin antigen prepared further can be used to immunize to obtain anti-lorcaserin antibodies with high cost-effectiveness, high sensitivity and strong specificity, and the polyclonal antibodies obtained by immunizing New Zealand white rabbits can be used for rapid and accurate immunodetection and analysis of lorcaserin. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the liquid chromatogram of lorcaserin artificial hapten I.

[0047] Figure 2 This is the mass spectrum of lorcaserin artificial hapten I.

[0048] Figure 3 These are ultraviolet scanning images of bovine serum albumin, lorcaserin artificial hapten I, lorcaserin artificial hapten VI, and lorcaserin artificial antigen II. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0050] In the embodiment of the present invention, the formula of MOP developing agent is as follows: 95% ethanol, dichloromethane, 1,4-dioxane, and concentrated ammonia in a volume ratio of 8:10:1:1.

[0051] Example 1 This embodiment provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen II and a preparation method thereof.

[0052] A method for preparing a lorcaserin artificial antigen comprises the following steps: (1) Preparation of artificial hapten I of lorcaserin: (S.1) 116 mg (0.5 mmol) of lorcaserin hydrochloride was dissolved in 15 mL of deionized water and the pH was adjusted to 9 with aqueous ammonia. A large amount of white precipitate was produced in the solution. The solution was filtered to obtain an alkaline solution. (S.2) The alkaline solution obtained in step (S.1) was extracted three times with 15 mL of dichloromethane each time, and the dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 96 mg (0.491 mmol) of colorless oil A; (S.3) The colorless oil A obtained in step (S.2) was dissolved in 9.6 mL of acetone in a 50 mL round-bottom flask, and 271 mg (1.963 mmol) of potassium carbonate was added, and the mixture was stirred at 25° C. for 0.5 h. Then, 107 μL (0.982 mmol) of ethyl bromoacetate was added, and the mixture was stirred at 25° C. for 1.5 h. TLC detection (developing solvent: ethyl acetate: ammonia water = 10 mL: 3 drops) showed that the reaction was complete, Rf = 0.7. The mixture was filtered and dried to obtain 112 mg (0.398 mmol) of colorless oil B; (S.4) Prepare 1 mol / L NaOH solution: dissolve 8 g NaOH in 200 mL deionized water to obtain 1 mol / L NaOH solution; prepare 1 mol / L hydrochloric acid solution: dilute 16.95 mL of 37% hydrochloric acid with deionized water to 200 mL to obtain 1 mol / L hydrochloric acid solution. Then the colorless oil B obtained in step (S.3) was dissolved in a 50 mL single-necked flask with 2.26 mL of tetrahydrofuran and 2.59 mL of methanol, and 11.2 mL of a prepared 1 mol / L NaOH solution was added. At this time, a large amount of precipitate was produced, and the solution was a milky white suspension. The reaction was stirred at 25°C for 3 hours. TLC detection (developing agent was MOP) showed that the reaction was basically complete, and the product Rf = 0.3-0.4. The pH was adjusted to 3 with a prepared 1 mol / L hydrochloric acid solution, and the mixture was directly dried. 5 mL of anhydrous ethanol was used for extraction each time, and the extraction was repeated 3 times. The mixture was filtered and dried. After separation and purification by TLC (developing agent was MOP, and the solvent and eluent were anhydrous ethanol), 95 mg (0.375 mmol) of lorcaserin artificial hapten I was obtained.

[0053] The molecular structure of lorcaserin artificial hapten I is shown in formula (I):

[0054] The liquid chromatogram of the artificial hapten Ⅰ of lorcaserin in this example is as follows Figure 1 The mass spectrum of the artificial hapten I of lorcaserin in this example is shown in Figure 2 shown.

[0055] from Figure 1 It can be seen that the purity of the purified lorcaserin artificial hapten I reaches more than 99%. Figure 2It can be seen that the characteristic peaks of the lorcaserin artificial hapten I obtained in this example are 254.09, 276.08, 277.08, and 298.11, respectively, which are consistent with its theoretical relative molecular mass of 253. It can be determined that the final compound obtained in step (S.4) is the lorcaserin artificial hapten I designed by the present invention.

[0056] (2) Preparation of lorcaserin artificial antigen II: (a) In a 50 mL single-necked round-bottom flask, 95 mg (0.375 mmol) of lorcaserin artificial hapten I obtained in step (S.4) was completely dissolved in 4.75 mL of N,N-dimethylformamide (DMF), and 58 mg (0.504 mmol) of N-hydroxysuccinimide and 103.3 mg (0.504 mmol) of cyclohexylcarbodiimide were added, and the mixture was stirred at 25° C. for 18 h to terminate the reaction. The reaction product was centrifuged and the supernatant was collected for later use; (b) Weigh 14.5g (40.503mmol) disodium hydrogen phosphate dodecahydrate, 43.875g (750mmol) sodium chloride, and 1.495g (9.583mmol) sodium dihydrogen phosphate dihydrate, dissolve in deionized water to 5.0L, and obtain 0.01M PBS buffer with a pH of 7.4. Weigh 237.5mg bovine serum albumin and dissolve in 47.5mL prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 5mg / mL. Under rapid stirring, slowly add the supernatant obtained in step (a) to the prepared bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution is 1:10, and the obtained mixture is stored at 4°C overnight to obtain an artificial antigen mixture. The artificial antigen mixture is transferred into a dialysis bag, dialyzed 7 times with the above-prepared PBS buffer, centrifuged after the dialysis, and the supernatant is taken to obtain lorcaserin artificial antigen II.

[0057] The molecular structure of lorcaserin artificial antigen II is shown in formula (II): Among them, BSA is bovine serum albumin, and n is 55-60.

[0058] The synthetic route of lorcaserin artificial antigen II in this embodiment is as follows: Among them, MeOH represents anhydrous methanol, THF represents tetrahydrofuran, DMF represents N,N-dimethylformamide, and DCC represents cyclohexylcarbodiimide. Since BSA and BGG are both globulins, they are represented by circles in the figure. The haptens are very small compared to the carrier protein, but for the convenience of expression, the haptens are not drawn very small. At this time, n≈57. The UV scanning images of bovine serum albumin, lorcaserin artificial hapten I, and lorcaserin artificial antigen II are shown in Figure 1. Figure 3 shown.

[0059] Figure 3 In the figure, curve a is the UV scanning graph of lorcaserin artificial hapten I, curve c is the UV scanning graph of lorcaserin artificial antigen II, and curve d is the UV scanning graph of bovine serum albumin. The maximum absorption wavelength of lorcaserin artificial hapten I is 248 nm, and the maximum absorption wavelength of lorcaserin artificial antigen II is 355 nm. Compared with lorcaserin artificial hapten I and bovine serum albumin, the maximum absorption wavelength of lorcaserin artificial antigen II changes significantly, indicating that lorcaserin artificial hapten I is successfully coupled with bovine serum albumin.

[0060] Example 2 The difference between this embodiment and embodiment 1 is that: This embodiment provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen II and a preparation method thereof.

[0061] A method for preparing a lorcaserin artificial antigen, wherein: (1) Preparation process of artificial hapten I of lorcaserin: step (S.1) adjusting pH to 8 with aqueous ammonia; step (S.3) dissolving 96 mg (0.491 mmol) of colorless oil A obtained in step (S.2) in 5.33 mL of acetone, adding 237 mg (1.718 mmol) of potassium carbonate; and then adding 81.6 μL (0.736 mmol) of ethyl bromoacetate; step (S.4) preparing 0.5 mol / L NaOH solution: dissolving 4 g of NaOH in 200 mL of deionized water to obtain 0.5 mol / L NaOH solution; preparing 0.5 mol / L hydrochloric acid solution: diluting 8.475 mL of 37% hydrochloric acid to 200 mL with deionized water to obtain 0.5 mol / L hydrochloric acid solution. Then, 108 mg (0.384 mmol) of the colorless oil B obtained in step (S.3) was dissolved in 1.8 mL of tetrahydrofuran and 2.4 mL of methanol in a 50 mL single-necked flask, and 9.6 mL of a prepared 0.5 mol / L NaOH solution was added; the pH was adjusted to 2 with a prepared 0.5 mol / L hydrochloric acid solution; the rest was the same as in Example 1. Finally, lorcaserin artificial hapten I was obtained.

[0062] (2) Preparation process of lorcaserin artificial antigen II: In step (a), 92 mg (0.366 mmol) of lorcaserin artificial hapten I obtained in step (S.4) was completely dissolved in 3.06 mL of N,N-dimethylformamide (DMF), 59 mg (0.512 mmol) of N-hydroxysuccinimide and 105 mg (0.512 mmol) of cyclohexylcarbodiimide were added, and the mixture was stirred at 20° C. for 20 h; in step (b), 150 mg of bovine serum albumin was weighed and dissolved in 37.4 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 4 mg / mL; the volume ratio of the supernatant to the bovine serum albumin solution was 0.9:11, and the obtained mixed solution was stored at 3° C. overnight; the rest was the same as in Example 1. Finally, lorcaserin artificial antigen II was obtained.

[0063] Example 3 The difference between this embodiment and embodiment 1 is that: This embodiment provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen II and a preparation method thereof.

[0064] A method for preparing a lorcaserin artificial antigen, wherein: (1) Preparation process of lorcaserin artificial hapten I: step (S.1) adjusting pH to 10 with aqueous ammonia; step (S.3) dissolving 96 mg (0.491 mmol) of colorless oil A obtained in step (S.2) in 13 mL of acetone, adding 305 mg (2.209 mmol) of potassium carbonate; and then adding 136 μL (1.227 mmol) of ethyl bromoacetate; step (S.4) preparing 2 mol / L NaOH solution: dissolving 16 g of NaOH in 200 mL of deionized water to obtain a 2 mol / L NaOH solution; preparing 1.5 mol / L hydrochloric acid solution: diluting 25.425 mL of 37% hydrochloric acid to 200 mL with deionized water to obtain a 1.5 mol / L hydrochloric acid solution. Then, 137 mg (0.487 mmol) of the colorless oil B obtained in step (S.3) was dissolved in 3.73 mL of tetrahydrofuran and 3.73 mL of methanol in a 50 mL single-necked flask, and 14.95 mL of a prepared 2 mol / L NaOH solution was added; the pH was adjusted to 4 with a prepared 1.5 mol / L hydrochloric acid solution; the rest was the same as in Example 1. Finally, lorcaserin artificial hapten I was obtained.

[0065] (2) Preparation process of lorcaserin artificial antigen II: In step (a), 118 mg (0.466 mmol) of lorcaserin artificial hapten I obtained in step (S.4) was completely dissolved in 7.08 mL of N,N-dimethylformamide (DMF), 80.38 mg (0.699 mmol) of N-hydroxysuccinimide and 143.29 mg (0.699 mmol) of cyclohexylcarbodiimide were added, and the mixture was stirred at 30°C for 19 hours; in step (b), 348 mg of bovine serum albumin was weighed and dissolved in 58 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 6 mg / mL; the volume ratio of the supernatant to the bovine serum albumin solution was 1.1:9, and the obtained mixed solution was stored at 5°C overnight; the rest was the same as in Example 1. Finally, lorcaserin artificial antigen II was obtained.

[0066] Comparative Example 1 The difference between this comparative example and Example 1 is: This comparative example provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen III and a preparation method thereof.

[0067] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten I is the same as that in Example 1. (2) the preparation process of artificial lorcaserin antigen III: in step (b), the volume ratio of supernatant to bovine serum albumin solution is replaced by a volume ratio of supernatant to bovine serum albumin solution of 1:5 with a volume ratio of supernatant to bovine serum albumin solution of 1:10. The rest is the same as that in Example 1. Finally, artificial lorcaserin antigen III is obtained. The synthetic route of artificial lorcaserin antigen III in this comparative example is the same as that in Example 1, and at this time n≈42.

[0068] Comparative Example 2 The difference between this comparative example and Example 1 is: This comparative example provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen IV and a preparation method thereof.

[0069] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten I is the same as that in Example 1. (2) the preparation process of artificial lorcaserin antigen IV: in step (b), bovine serum albumin (i.e., carrier protein) is replaced with bovine immunoglobulin gamma globulin, and coupled with lorcaserin hapten I, using the same coupling steps as in Example 1, and the rest is the same as in Example 1. Finally, artificial lorcaserin antigen IV is obtained. The synthetic route of artificial lorcaserin antigen IV in this comparative example is as follows: Among them, BGG is bovine immunoglobulin G, and n≈18 in this case.

[0070] Comparative Example 3 The difference between this comparative example and comparative example 1 is: This comparative example provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen V and a preparation method thereof.

[0071] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten I is the same as that in Example 1. (2) the preparation process of artificial lorcaserin antigen V: in step (b), bovine serum albumin (i.e., carrier protein) is replaced with bovine immunoglobulin gamma globulin, and coupled with lorcaserin hapten I, and the same coupling steps as in Comparative Example 1 are adopted, and the rest are the same as in Comparative Example 1. Finally, artificial lorcaserin antigen V is obtained. The synthetic route of artificial lorcaserin antigen V in this comparative example is the same as that in Comparative Example 2, and in this case n≈15.

[0072] Comparative Example 4 This comparative example provides a lorcaserin artificial hapten VI and a preparation method thereof, and a lorcaserin artificial antigen VII and a preparation method thereof.

[0073] A method for preparing a lorcaserin artificial antigen comprises the following steps: (1) Preparation of lorcaserin artificial hapten VI: (S.1) Prepare a 10% sodium bicarbonate solution. Prepare a 1mol / L NaOH solution: dissolve 8g NaOH in 200mL deionized water to obtain a 1mol / L NaOH solution. Prepare a 1mol / L hydrochloric acid solution: dilute 16.95mL of 37% hydrochloric acid to 200mL with deionized water to obtain a 1mol / L hydrochloric acid solution. Weigh 100mg (0.473mmol) 7-hydroxychlorcaserin and dissolve it in 5mL deionized water in a 50mL round-bottom flask, place it in an ice bath, and slowly adjust the pH to 11 with the prepared 10% sodium bicarbonate solution to obtain an alkaline solution; (S.2) Weigh 191 mg (0.567 mmol) of 9-fluorenylmethyl-N-succinimidyl carbonate and dissolve it in 2.5 mL of 1,4-dioxane. Slowly add it dropwise to the alkaline solution obtained in step (S.1) under an ice bath. After adding, heat it to room temperature (25°C) and stir to react for 18 hours. After TLC detection (developing solvent is ethyl acetate), the reaction is complete. End the reaction, add 10 mL of deionized water, wash the aqueous phase with 5 mL of ether each time, wash 3 times, and collect the aqueous phase. The aqueous phase is adjusted to pH = 2 with 1 mol / L hydrochloric acid solution prepared in step (S.1), and a large amount of white precipitate is produced. It is extracted with 10 mL of ethyl acetate each time, and extracted 3 times. The ethyl acetate phases are combined, and the organic phase is washed with 10 mL of deionized water and 10 mL of saturated brine respectively. The ethyl acetate phase is collected, dried over anhydrous sodium sulfate, filtered, and dried to obtain 149 mg (0.344 mmol) of colorless oil A; (S.3) The colorless oil A obtained in step (S.2) was dissolved in 14.9 mL of acetone in a 50 mL round-bottom flask, and 188.5 mg (1.376 mmol) of potassium carbonate and 75.5 μL (0.688 mmol) of ethyl bromoacetate were added, and nitrogen was introduced for protection. The reaction was stirred at 25°C in the dark for 18 h. The reaction was complete by TLC detection (developing solvent was ethyl acetate: ammonia water = 6 mL: 3 drops), and the product point Rf = 0.5. The product was filtered and dried, and separated and purified by TLC (developing solvent was ethyl acetate: ammonia water = 6 mL: 3 drops, and the solvent and eluent were anhydrous ethanol) to obtain 159 mg (0.306 mmol) of colorless oil B; (S.4) The colorless oil B obtained in step (S.3) was dissolved in a 50 mL round-bottom flask with 3.22 mL of tetrahydrofuran and 3.68 mL of methanol. At this time, the solution was a colorless transparent liquid. 15.9 mL of the 1 mol / L NaOH solution prepared in step (S.1) was added, and the solution immediately turned white and turbid. The reaction was stirred at 25°C for 5 hours, and the solution became clear. The reaction was complete by TLC detection (developing agent was MOP), and the product point Rf = 0.3. The pH was adjusted to 4 with the 1 mol / L hydrochloric acid solution prepared in step (S.1), and a precipitate was generated. The extraction was performed with 10 mL of dichloromethane each time, and the extraction was performed 3 times. The dichloromethane phase was collected, dried, filtered, and dried to obtain 144 mg (0.293 mmol) of lorcaserin artificial hapten VI. The ultraviolet scanning image of lorcaserin artificial hapten VI is as follows: Figure 3 shown. Figure 3 In the figure, curve b is the ultraviolet scanning diagram of the artificial hapten VI of lorcaserin, and the maximum absorption wavelength of the artificial hapten VI of lorcaserin is 266 nm.

[0074] (2) Preparation of lorcaserin artificial antigen VII: (a) In a 50 mL single-necked round-bottom flask, 144 mg (0.293 mmol) of lorcaserin artificial hapten VI obtained in step (S.4) was completely dissolved in 7.2 mL of DMF, 45.5 mg (0.396 mmol) of N-hydroxysuccinimide and 81.6 mg (0.396 mmol) of cyclohexylcarbodiimide were added, and the mixture was stirred at 25° C. for 18 h. The reaction was terminated, and the reaction product was centrifuged to collect the supernatant for later use; (b) Weigh 14.5g (40.503mmol) disodium hydrogen phosphate dodecahydrate, 43.875g (750mmol) sodium chloride, and 1.495g (9.583mmol) sodium dihydrogen phosphate dihydrate, dissolve in deionized water to 5.0L, and obtain 0.01M PBS buffer with a pH of 7.4. Weigh 360mg bovine serum albumin and dissolve in 72mL of the prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 5mg / mL. Under rapid stirring, slowly add the supernatant obtained in step (a) to the prepared bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution is 1:10, and the obtained mixture is stored at 4°C overnight to obtain an artificial antigen mixture. The artificial antigen mixture is transferred into a dialysis bag, dialyzed 7 times with the above-prepared PBS buffer, centrifuged after the dialysis, and the supernatant is taken to obtain the lorcaserin artificial antigen VII.

[0075] The synthetic route of lorcaserin artificial antigen VII in this comparative example is as follows: Wherein Fmoc-Osu represents 9-fluorenylmethyl-N-succinimidyl carbonate, and n≈17 in this case.

[0076] Comparative Example 5 The difference between this comparative example and comparative example 4 is that: This comparative example provides a lorcaserin artificial hapten VI and a preparation method thereof, and a lorcaserin artificial antigen VIII and a preparation method thereof.

[0077] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten VI is the same as that in comparative example 4. (2) the preparation process of artificial lorcaserin antigen VIII: in step (b), the volume ratio of supernatant to bovine serum albumin solution is replaced with a volume ratio of supernatant to bovine serum albumin solution of 1:5, and the rest is the same as that in comparative example 4. Finally, artificial lorcaserin antigen VIII is obtained. The synthetic route of artificial lorcaserin antigen VIII in this comparative example is the same as that in comparative example 4, and in this case n≈13.

[0078] Comparative Example 6 The difference between this comparative example and comparative example 4 is that: This comparative example provides a lorcaserin artificial hapten VI and a preparation method thereof, and a lorcaserin artificial antigen IX and a preparation method thereof.

[0079] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten VI is the same as that in comparative example 4. (2) the preparation process of artificial lorcaserin antigen IX: in step (b), bovine serum albumin (i.e., carrier protein) is replaced with bovine immunoglobulin gamma globulin, and coupled with lorcaserin hapten VI, the same coupling steps as those in comparative example 4 are adopted, and the rest are the same as those in comparative example 4. Finally, artificial lorcaserin antigen IX is obtained.

[0080] The synthetic route of lorcaserin artificial antigen IX in this comparative example is as follows: At this time n≈7.

[0081] Comparative Example 7 The difference between this comparative example and comparative example 4 is that: This comparative example provides a lorcaserin artificial hapten VI and a preparation method thereof, and a lorcaserin artificial antigen X and a preparation method thereof.

[0082] A method for preparing an artificial lorcaserin antigen, wherein: (1) the preparation process of artificial lorcaserin hapten VI is the same as that in Comparative Example 4. (2) the preparation process of artificial lorcaserin antigen IX: in step (b), bovine serum albumin (i.e., carrier protein) is replaced with bovine immunoglobulin gamma globulin, and coupled with lorcaserin hapten VI, using the same coupling steps as in Comparative Example 5, and the rest is the same as in Comparative Example 4. Finally, artificial lorcaserin antigen X is obtained. The synthetic route of artificial lorcaserin antigen X in this comparative example is the same as that in Comparative Example 6, and in this case n≈4.

[0083]

Performance determination of lorcaserin artificial antigen

[0084] Table 1: Calculation table of molar absorption coefficients of artificial haptens I and VI of lorcaserin

[0085] Determination of protein concentration of conjugate: prepare lorcaserin artificial antigen according to the methods in Example 1 and Comparative Examples 1 to 7. Prepare 1 mL of bovine serum albumin solution with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL with PBS buffer, add 3 mL of Coomassie Brilliant Blue staining solution, mix immediately, warm in a 30°C water bath for 5 min, make parallel samples for each concentration, measure the absorbance at 655 nm, and draw a curve of the relationship between protein concentration and absorbance. Dilute the artificial antigen solution (prepared with PBS buffer) in a certain proportion, measure the absorbance of the artificial antigen at 655 nm, read the corresponding protein concentration value of the artificial antigen solution from the curve, and the calculated data results are shown in Table 2 below.

[0086] Coupling ratio determination: When the conjugate hapten is lorcaserin artificial hapten VI: prepare 100 μg / mL bovine serum albumin PBS solution and 100 μg / mL bovine immunoglobulin G PBS solution, dilute the conjugate (i.e. lorcaserin artificial antigen) to 100 μg / mL with PBS, measure the absorbance value A1 at 266 nm, and measure the absorbance value A2 (selected according to the protein conjugated to the lorcaserin artificial antigen) with PBS as blank. The calculation formula of the coupling ratio γ is: γ=[(A1-A2) / ε] / (100×10 -3 / 66400), where ε is the molar absorption coefficient (L / mol), 66400 is the molecular weight of bovine serum albumin, and 100×10 -3 is the concentration of bovine serum albumin (g / L). The calculated data results are shown in Table 2 below.

[0087] When bovine immunoglobulin G is used as the carrier, the calculation formula for the coupling ratio γ is: γ = [(A1-A2) / ε] / (100×10 -3 / 43000), where 43000 is the molecular weight of bovine immunoglobulin G, 100×10 -3 is the concentration of bovine immunoglobulin (g / L). The calculated data results are shown in Table 2 below.

[0088] Table 2: Calculation data of coupling ratio of each lorcaserin artificial antigen and protein concentration of the conjugate

[0089] As shown in Tables 1 and 2, the structure of the artificial hapten, the volume ratio between the artificial hapten and the carrier protein solution when coupled, and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten and the carrier protein are cross-linked.

[0090] (2) Animal immunization The artificial lorcaserin antigens were prepared according to the methods of Example 1 and Comparative Examples 1 to 7. New Zealand white rabbits were immunized with the prepared artificial lorcaserin antigens, and the titers of the obtained immune sera were tested by ELISA. The test results are shown in Table 3 below.

[0091] Table 3: Titer test results of each immune serum serial number Lorcaserin artificial antigen Immune serum titer Example 1 II 1：256000 Comparative Example 1 III 1：64000 Comparative Example 2 IV 1：16000 Comparative Example 3 V 1：8000 Comparative Example 4 VII 1：16000 Comparative Example 5 VIII 1：8000 Comparative Example 6 IX 1：4000 Comparative Example 7 X / .

[0092] As shown in Table 3, compared with Example 1, the titers of the immune serum obtained by immunizing animals with the artificial lorcaserin antigen prepared by the methods in the comparative examples were all low and could not be used in immunoassays. However, the titer of the immune serum obtained by immunizing animals with the artificial lorcaserin antigen II prepared by the method in Example 1 reached 1:256000, which could be fully used in immunoassays, and could provide a more convenient, rapid and accurate way for the detection of lorcaserin.

[0093] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A lorcaserin artificial hapten, characterized in that: Its molecular structure is shown in formula (I):

2. The method for preparing a lorcaserin artificial hapten according to claim 1, characterized in that: The following steps are involved: (S.1) dissolving lorcaserin hydrochloride in deionized water, and adjusting the pH to alkaline with aqueous ammonia to obtain an alkaline solution; (S.2) extracting the alkaline solution obtained in step (S.1) with dichloromethane, collecting the dichloromethane phase, drying, filtering, and drying to obtain a colorless oil A; (S.3) dissolving the colorless oil A obtained in step (S.2) in a solvent, adding potassium carbonate, and then adding ethyl bromoacetate, mixing evenly and heating and stirring to react, filtering after the reaction is complete, drying to obtain a colorless oil B; (S.4) Prepare sodium hydroxide solution and hydrochloric acid solution respectively, then add tetrahydrofuran, methanol and the prepared sodium hydroxide solution to the colorless oil B obtained in step (S.3), heat and stir to react until the reaction is complete, adjust the pH to acidic with the prepared hydrochloric acid solution, dry it, extract with anhydrous ethanol, filter, dry it, separate, and obtain lorcaserin artificial hapten I.

3. The method for preparing a lorcaserin artificial hapten according to claim 2, characterized in that: The pH of the alkaline solution in step (S.1) is 8 to 10; The molar ratio of the colorless oil A added in step (S.3) to potassium carbonate is 1:(3.5-4.5); the molar ratio of the colorless oil A to ethyl bromoacetate is 1:(1.5-2.5); the solvent is any one of acetone, N,N-dimethylformamide, and anhydrous ethanol; the solid-liquid ratio of the colorless oil A to the solvent is 9-11:0.5-1.5 mg / mL.

4. The method for preparing a lorcaserin artificial hapten according to claim 2, characterized in that: The solid-liquid ratio of the colorless oil B, tetrahydrofuran, methanol and sodium hydroxide solution added in step (S.4) is 90-110:1.5-3:2-3:8-12 mg / mL / mL / mL; the concentration of the prepared sodium hydroxide solution is 0.5-2 mol / L; the concentration of the prepared hydrochloric acid solution is 0.5-1.5 mol / L; and the pH is adjusted to 2-4 with the prepared hydrochloric acid solution.

5. A lorcaserin artificial antigen, characterized in that: Its molecular structure is shown in formula (II): Among them, BSA is bovine serum albumin, and n is 55-60.

6. The method for preparing a lorcaserin artificial antigen according to claim 5, characterized in that: The method comprises the following steps: coupling the lorcaserin artificial hapten I as claimed in claim 1 with bovine serum albumin by using the N-hydroxysuccinimide active ester method, and obtaining the lorcaserin artificial antigen II after purification.

7. The method for preparing a lorcaserin artificial antigen according to claim 6, characterized in that: The following steps are involved: (a) Mixing lorcaserin artificial hapten I, N-hydroxysuccinimide and cyclohexylcarbodiimide in N,N-dimethylformamide, heating and stirring until the reaction is complete, centrifuging and collecting the supernatant; (b) adding the supernatant obtained in step (a) to the bovine serum albumin solution, mixing well and standing overnight, dialyzing and centrifuging to obtain the supernatant to obtain lorcaserin artificial antigen II.

8. The method for preparing a lorcaserin artificial antigen according to claim 7, characterized in that: The molar ratio of the lorcaserin artificial hapten, N-hydroxysuccinimide and cyclohexylcarbodiimide added in step (a) is 1:(1.35-1.5):(1.35-1.5); the solid-liquid ratio of the lorcaserin artificial hapten and N,N-dimethylformamide is 15-25:0.5-1.5 mg / mL; the reaction temperature of the heating and stirring reaction process is 20-30° C., and the reaction time is 18-20 hours; The volume ratio of the supernatant added in step (b) to the bovine serum albumin solution is 0.9-1.1:9-11; the concentration of the bovine serum albumin solution is 4-6 mg / mL; and the temperature of standing overnight is 3-5°C.

9. Use of the artificial lorcaserin antigen as claimed in claim 5 in preparing anti-lorcaserin antibodies.

10. An anti-lorcaserin artificial antibody, characterized in that: The artificial lorcaserin antigen according to claim 5 is obtained by animal immunization and can generate a specific immune reaction with lorcaserin.

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