A lorcaserin artificial hapten, artificial antigen, and preparation method and application thereof

Abstract

The present application belongs to the technical field of bio-chemical industry, and particularly relates to a lorcaserin artificial hapten, a lorcaserin artificial antigen, and a preparation method and application thereof. The lorcaserin hydrochloride is used as a starting material for synthesizing the artificial hapten, the lorcaserin artificial hapten of the present application retains the characteristic structure of lorcaserin to the greatest extent, and has an active group which can be coupled with a carrier protein, and can be used as an antigenic determinant; the lorcaserin artificial antigen prepared further can be used for immunization to obtain an anti-lorcaserin artificial antibody with high affinity, high sensitivity and high specificity, the titer of the immune serum obtained by immunizing a New Zealand white rabbit is as high as 1:256000, and the immune serum can be used for rapid and accurate immunodetection and immunological analysis of lorcaserin.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological chemical industry, and particularly relates to a lorcaserin artificial hapten, a lorcaserin artificial antigen, and a preparation method and application thereof. BACKGROUND

[0002] Lorcaserin is a novel weight loss drug acting on the central nervous system, which reduces the amount of food intake and promotes satiety by selectively stimulating the hypothalamic satiety center, and is generally used for treating binge eating disorder and obesity. However, due to its many and significant side effects, the general weight loss population should not use it at will. If it is necessary to use these drugs, it must be used under the guidance of a doctor and self-purchase should be avoided to prevent serious consequences. The adverse reactions mainly include headache, dizziness, fatigue, nausea, dry mouth, constipation, etc.

[0003] Lorcaserin has a certain impact on the cardiovascular system, and therefore has a carcinogenic risk for patients who already have cardiovascular diseases such as heart valve lesions, arrhythmia, etc., and patients who have a risk of cardiovascular diseases. Excessive abuse brings pain such as kidney necrosis, hemodialysis, abnormal liver function, and all-day wearing of paper diapers.

[0004] At present, high-performance liquid chromatography, gas chromatography, mass spectrometry, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, infrared spectrum, etc. are mainly used for analysis and determination in China. However, due to the high cost of use and maintenance, they are not suitable for large-scale sampling analysis. Therefore, it is necessary to synthesize a lorcaserin artificial hapten and artificial antigen to meet the needs of immune analysis. At present, there is still no related report on lorcaserin artificial hapten and artificial antigen.

[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 lorcaserin artificial antigen is the basis for the realization of the method. SUMMARY

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

[0007] To achieve the above-mentioned application purposes, the application realizes the following technical solutions:

[0008] A lorcaserin artificial hapten, the molecular structure formula of which is shown as formula (I):

[0009]

[0010] Formula (I).

[0011] A preparation method of a lorcaserin artificial hapten as described above comprises the following steps:

[0012] (S.1) dissolving lorcaserin hydrochloride in deionized water, adjusting pH to alkaline with ammonia water to obtain an alkaline solution;

[0013] (S.2) extracting the alkaline solution obtained in step (S.1) with dichloromethane, collecting dichloromethane phase, drying, filtering, and drying to obtain colorless oil A;

[0014] (S.3) dissolving colorless oil A obtained in step (S.2) in a solvent, adding potassium carbonate, then adding ethyl bromoacetate, uniformly mixing and heating and stirring to react, filtering after the reaction is completed, drying, and obtaining colorless oil B;

[0015] (S.4) respectively preparing sodium hydroxide solution and hydrochloric acid solution, then adding tetrahydrofuran, methanol, and the prepared sodium hydroxide solution to colorless oil B obtained in step (S.3), heating and stirring to react until the reaction is completed, adjusting pH to acid with the prepared hydrochloric acid solution, drying, extracting with anhydrous ethanol, filtering, drying, separating, and obtaining lorcaserin artificial hapten I.

[0016] The present application uses lorcaserin hydrochloride as a starting material for synthesizing lorcaserin artificial hapten, which not only maximally retains the characteristic structure of lorcaserin, but also has an active group that can be coupled with carrier proteins, and can be used as an antigenic determinant. The present application introduces a connecting arm at the imino group of lorcaserin, which maximally retains the characteristic structure of lorcaserin. Compared with a cyclic connecting arm, the present application uses a straight chain as a connecting arm, which can reduce non-specific binding in immunoassay, and also can reduce the immune response to the connecting arm in immunization, thereby improving the probability of producing specific antibodies.

[0017] As preferred, the pH of the alkaline solution in step (S.1) is 8-10.

[0018] As further preferred, the pH of the alkaline solution in step (S.1) is 9.

[0019] As preferred, the molar ratio of colorless oil A to potassium carbonate added in step (S.3) is 1:(3.5-4.5).

[0020] As further preferred, the molar ratio of colorless oil A to potassium carbonate added in step (S.3) is 1:4.

[0021] As preferred, the molar ratio of colorless oil A to ethyl bromoacetate added in step (S.3) is 1:(1.5-2.5).

[0022] As further preferred, the molar ratio of the colorless oil A added in step (S.3) to ethyl bromoacetate is 1:2.

[0023] As preferred, the solvent in step (S.3) is any one of acetone, N,N-dimethylformamide, anhydrous ethanol.

[0024] As preferred, the feed liquid ratio of the colorless oil A added in step (S.3) to the solvent is 9-11:0.5-1.5 mg / mL.

[0025] As further preferred, the feed liquid ratio of the colorless oil A added in step (S.3) to the solvent is 10:1 mg / mL.

[0026] As preferred, the feed liquid ratio of the colorless oil B, tetrahydrofuran, methanol, sodium hydroxide solution added in step (S.4) is 90-110:1.5-3:2-3:8-12 mg / mL / mL / mL.

[0027] As further preferred, the feed liquid ratio of the colorless oil B, tetrahydrofuran, methanol, sodium hydroxide solution added in step (S.4) is 100:2.02:2.31:10 mg / mL / mL / mL.

[0028] As preferred, the concentration of the prepared sodium hydroxide solution in step (S.4) is 0.5-2 mol / L; the concentration of the prepared hydrochloric acid solution is 0.5-1.5 mol / L.

[0029] As further preferred, the concentration of the prepared sodium hydroxide solution in step (S.4) is 1 mol / L; the concentration of the prepared hydrochloric acid solution is 1 mol / L.

[0030] As preferred, the pH in step (S.4) is adjusted to 2-4 with the prepared hydrochloric acid solution.

[0031] As further preferred, the pH in step (S.4) is adjusted to 3 with the prepared hydrochloric acid solution.

[0032] A lorcaserin artificial antigen, the molecular structural formula of which is shown in formula (II):

[0033]

[0034] wherein BSA is bovine serum albumin, and n is 55-60. Preferably, n is 57.

[0035] A preparation method of a lorcaserin artificial antigen as described above, comprising the following steps:

[0036] The chlorocarpaline artificial hapten I is coupled with bovine serum albumin by using N-hydroxy succinimide active ester method, and the chlorocarpaline artificial antigen II is obtained after purification.

[0037] As preferred, the preparation method of the chlorocarpaline artificial antigen as described above comprises the following steps:

[0038] (a) mixing the chlorocarpaline artificial hapten I, N-hydroxy succinimide and cyclohexyl carbodiimide in N,N-dimethylformamide, heating and stirring to react until the reaction is complete, centrifuging, and taking the supernatant;

[0039] (b) adding the supernatant obtained in step (a) into a bovine serum albumin solution, mixing uniformly and standing overnight, dialyzing, centrifuging to take the supernatant, and obtaining the chlorocarpaline artificial antigen II.

[0040] As preferred, the molar ratio of the chlorocarpaline artificial hapten, N-hydroxy succinimide and cyclohexyl carbodiimide added in step (a) is 1: (1.35-1.5): (1.35-1.5).

[0041] As further preferred, the molar ratio of the chlorocarpaline artificial hapten, N-hydroxy succinimide and cyclohexyl carbodiimide added in step (a) is 1:1.35:1.35.

[0042] As preferred, the ratio of the chlorocarpaline artificial hapten to N,N-dimethylformamide added in step (a) is 15-25:0.5-1.5 mg / mL.

[0043] As further preferred, the ratio of the chlorocarpaline artificial hapten to N,N-dimethylformamide added in step (a) is 20:1 mg / mL.

[0044] As preferred, the reaction temperature in the heating and stirring reaction process in step (a) is 20-30°C, and the reaction time is 18-20 h.

[0045] As further preferred, the reaction temperature in the heating and stirring reaction process in step (a) is 25°C, and the reaction time is 18 h.

[0046] As preferred, the volume ratio of the supernatant to the bovine serum albumin solution added in step (b) is 0.9-1.1:9-11.

[0047] As further preferred, the volume ratio of the supernatant to the bovine serum albumin solution added in step (b) is 1:10.

[0048] As preferred, the concentration of the bovine serum albumin solution added in step (b) is 4-6 mg / mL.

[0049] As a further preference, the concentration of the bovine serum albumin solution added in step (b) is 5 mg / mL.

[0050] As a further preference, the method for preparing the bovine serum albumin solution comprises the following steps:

[0051] The bovine serum albumin is dissolved in a 0.01M PBS buffer solution, and the pH of the PBS buffer solution is 7.2-7.4.

[0052] The bovine serum albumin (BSA) is selected as the macromolecular carrier in the present application, and compared with bovine gamma globulin (BGG), the bovine serum albumin has the following advantages:

[0053] ①The bovine serum albumin can better and more combine with the lorcaserin artificial hapten, so as to prepare lorcaserin artificial antigens with different coupling ratios;

[0054] ②From the experiment, the stability of the lorcaserin artificial hapten after being combined with the bovine serum albumin is stronger, and it can be known from the seven-day stability evaluation that, under the same protein concentration, the bovine serum albumin combined with the lorcaserin artificial hapten does not have protein precipitation phenomenon, and is more stable; while the bovine gamma globulin combined with the lorcaserin artificial hapten produces a large amount of protein precipitation after the stability evaluation, so the bovine gamma globulin combined with the lorcaserin artificial hapten is easy to separate in the subsequent processing, so that the finally prepared artificial antigen is easy to produce precipitation in long-term storage, and has poor stability. The combination of the bovine serum albumin and the lorcaserin artificial hapten can be stored for a long time under different pH and temperature, and the protein is not easy to separate, and has more stability;

[0055] ③After the bovine serum albumin is combined with the lorcaserin artificial hapten, the specificity is stronger compared with the bovine gamma globulin;

[0056] ④The bovine serum albumin is used in the present application, which is cheaper and easier to obtain compared with the bovine gamma globulin, and can reduce the cost to a certain extent.

[0057] As a preference, the temperature for standing overnight in step (b) is 3-5℃.

[0058] As a further preference, the temperature for standing overnight in step (b) is 4℃.

[0059] The use of the lorcaserin artificial antigen as described above in the preparation of an anti-lorcaserin antibody.

[0060] An anti-lorcaserin artificial antibody is obtained by immunizing animals with the lorcaserin artificial antigen as described above, and can specifically immunoreact with lorcaserin.

[0061] The present application is found through repeated experiments that the titer of the immune serum obtained by immunizing New Zealand white rabbits with the lorcaserin artificial antigen is 1:256000. Therefore, it is shown that the lorcaserin artificial antigen of the present application can be used to immunize to obtain the anti-lorcaserin antibody with high affinity, high sensitivity and strong specificity, and the anti-lorcaserin antibody can be used for the immunodetection and analysis of lorcaserin.

[0062] Therefore, the present application has the following beneficial effects:

[0063] The lorcaserin artificial hapten of the present application retains the characteristic structure of lorcaserin to the greatest extent, and has an active group that can be coupled with a carrier protein, which can be used as an antigenic determinant; the lorcaserin artificial antigen prepared therefrom can be used to immunize to obtain the anti-lorcaserin antibody with high titer, high sensitivity and strong specificity, and the polyclonal antibody obtained by immunizing New Zealand white rabbits can be used for rapid and accurate immunodetection and analysis of lorcaserin. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 The liquid chromatogram of lorcaserin artificial hapten I.

[0065] Figure 2 The mass spectrum of lorcaserin artificial hapten I.

[0066] Figure 3 The ultraviolet scanning diagram of bovine serum albumin, lorcaserin artificial hapten I, lorcaserin artificial hapten VI, and lorcaserin artificial antigen II. DETAILED DESCRIPTION

[0067] The present application will be further described below in conjunction with the drawings and specific embodiments in the specification. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only a part of the embodiments of the present application, not all the embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor shall fall within the scope of protection of the present application.

[0068] In the embodiments of the present application, the formula of the MOP developing agent is as follows:

[0069] 95% ethanol, dichloromethane, 1,4-dioxane, concentrated ammonia water in a volume ratio of 8:10:1:1.

[0070] Example 1

[0071] The present embodiment provides a lorcaserin artificial hapten I and a preparation method thereof, and a lorcaserin artificial antigen II and a preparation method thereof.

[0072] A preparation method of lorcaserin artificial antigen, comprising the following steps:

[0073] (1) Preparation of lorcaserin artificial hapten I:

[0074] (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 ammonia water, a large amount of white precipitate was generated in the solution, and the alkaline solution was obtained by filtration;

[0075] (S.2) The alkaline solution obtained in step (S.1) was extracted with 15 mL of dichloromethane each time, and the extraction was performed for 3 times, the dichloromethane phase was collected, dried with anhydrous magnesium sulfate, filtered, and dried to obtain 96 mg (0.491 mmol) of colorless oil A;

[0076] (S.3) In a 50 mL round-bottom flask, the colorless oil A obtained in step (S.2) was dissolved in 9.6 mL of acetone, 271 mg (1.963 mmol) of potassium carbonate was added, and stirred at 25°C for 0.5 h, then 107 μL (0.982 mmol) of ethyl bromoacetate was added, and stirred at 25°C for 1.5 h, TLC detection (developing agent is ethyl acetate:ammonia water = 10 mL:3 drops) showed that the reaction was complete, Rf = 0.7, filtered, and dried to obtain 112 mg (0.398 mmol) of colorless oil B;

[0077] (S.4) Preparation of 1 mol / L NaOH solution: 8 g of NaOH was dissolved in 200 mL of deionized water to obtain 1 mol / L NaOH solution; preparation of 1 mol / L hydrochloric acid solution: 16.95 mL of 37% concentrated hydrochloric acid was diluted 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-neck flask with 2.26 mL of tetrahydrofuran and 2.59 mL of methanol, 11.2 mL of prepared 1 mol / L NaOH solution was added, at this time a large amount of precipitate was generated, the solution was a milky white suspension, and the reaction was stirred at 25°C for 3 h, TLC detection (developing agent is MOP) showed that the reaction was basically complete, the product Rf = 0.3-0.4, the pH was adjusted to 3 with the prepared 1 mol / L hydrochloric acid solution, and directly dried, 5 mL of anhydrous ethanol was used for extraction each time, the extraction was performed for 3 times, filtered, and dried, and after TLC (developing agent is MOP, solvent and eluent is anhydrous ethanol) separation and purification, 95 mg (0.375 mmol) of lorcaserin artificial hapten I was obtained.

[0078] The molecular structure of lorcaserin artificial hapten I is shown in formula (I):

[0079]

[0080] The liquid chromatogram of lorcaserin artificial hapten I in this embodiment is as follows: Figure 1 As shown. The mass spectrum of lorcaserin artificial hapten I in this embodiment is shown below. Figure 2 As shown.

[0081] from Figure 1 It can be seen that the purity of the purified lorcaserin artificial hapten I reached over 99%. From... Figure 2 As can be seen, the characteristic peaks of the lorcaserin artificial hapten I obtained in this embodiment are 254.09, 276.08, 277.08, and 298.11, 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 in this invention.

[0082] (2) Preparation of lorcaserin artificial antigen II:

[0083] (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). 58 mg (0.504 mmol) of N-hydroxysuccinimide and 103.3 mg (0.504 mmol) of cyclohexylcarbodiimide were added. The mixture was stirred at 25 °C for 18 h. The reaction was then stopped. The reaction product was centrifuged, and the supernatant was collected for later use.

[0084] (b) Weigh 14.5 g (40.503 mmol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (750 mmol) of sodium chloride, and 1.495 g (9.583 mmol) of sodium dihydrogen phosphate dihydrate, dissolve in deionized water, and bring the volume to 5.0 L to obtain a 0.01 M PBS buffer with a pH of 7.4. Weigh 237.5 mg of bovine serum albumin (BSA) and dissolve it in 47.5 mL of the prepared PBS buffer to obtain a BSA solution with a concentration of 5 mg / mL. Under rapid stirring, slowly add the supernatant obtained in step (a) to the prepared BSA solution at a volume ratio of 1:10. Incubate the resulting mixture at 4 °C overnight to obtain the artificial antigen mixture. Transfer the artificial antigen mixture to a dialysis bag and dialyze it 7 times with the prepared PBS buffer. After dialysis, centrifuge and collect the supernatant to obtain lorcaserin artificial antigen II.

[0085] The molecular structure of lorcaserin artificial antigen II is shown in formula (II):

[0086]

[0087] BSA is bovine serum albumin, and n is 55-60.

[0088] The synthetic route of the clonazepam artificial antigen II in this embodiment is shown as follows:

[0089]

[0090] In the figure, MeOH represents anhydrous methanol, THF represents tetrahydrofuran, DMF represents N,N-dimethylformamide, and DCC represents cyclohexyl carbodiimide. Since both BSA and BGG are globulins, they are represented by circles in the figure. The hapten is very small relative to the carrier protein, but it is not particularly small in the figure for ease of expression. At this time, n≈57. The ultraviolet scanning diagram of the bovine serum albumin, the clonazepam artificial hapten I, and the clonazepam artificial antigen II is shown in FIG. 2. Figure 3

[0091] Figure 3 In the figure, curve a is the ultraviolet scanning diagram of the clonazepam artificial hapten I, curve c is the ultraviolet scanning diagram of the clonazepam artificial antigen II, and curve d is the ultraviolet scanning diagram of the bovine serum albumin. The maximum absorption wavelength of the clonazepam artificial hapten I is 248 nm, and the maximum absorption wavelength of the clonazepam artificial antigen II is 355 nm. Compared with the clonazepam artificial hapten I and the bovine serum albumin, the maximum absorption wavelength of the clonazepam artificial antigen II changes obviously, indicating that the clonazepam artificial hapten I is coupled with the bovine serum albumin successfully.

[0092] Embodiment 2

[0093] The difference between this embodiment and embodiment 1 is that:

[0094] The embodiment provides a clonazepam artificial hapten I and a preparation method thereof and a clonazepam artificial antigen II and a preparation method thereof.

[0095] A preparation method of a clonazepam artificial antigen, wherein,

[0096] ​(1) The preparation process of the artificial hapten I of lorcaserin: step (S.1) adjust the pH to 8 with ammonia water; step (S.3) dissolve 96 mg (0.491 mmol) of the colorless oily substance A obtained in step (S.2) in 5.33 mL of acetone, and then add 237 mg (1.718 mmol) of potassium carbonate; further add 81.6 μL (0.736 mmol) of ethyl bromoacetate; step (S.4) prepare a 0.5 mol / L NaOH solution: dissolve 4 g of NaOH in 200 mL of deionized water to obtain a 0.5 mol / L NaOH solution; prepare a 0.5 mol / L hydrochloric acid solution: dilute 8.475 mL of 37% hydrochloric acid with deionized water to 200 mL to obtain a 0.5 mol / L hydrochloric acid solution. Then dissolve 108 mg (0.384 mmol) of the colorless oily substance B obtained in step (S.3) in 1.8 mL of tetrahydrofuran and 2.4 mL of methanol in a 50 mL single-necked flask, and then add 9.6 mL of the prepared 0.5 mol / L NaOH solution; adjust the pH to 2 with the prepared 0.5 mol / L hydrochloric acid solution; and the rest is the same as in Example 1. Finally, the artificial hapten I of lorcaserin is obtained.

[0097] (2) The preparation process of the artificial antigen II of lorcaserin:

[0098] In step (a), dissolve 92 mg (0.366 mmol) of the artificial hapten I of lorcaserin obtained in step (S.4) completely in 3.06 mL of N,N-dimethylformamide (DMF), and then add 59 mg (0.512 mmol) of N-hydroxysuccinimide and (105 mg (0.512 mmol) of cyclohexylcarbodiimide, and then stir the reaction at 20°C for 20 h; in step (b), weigh 150 mg of bovine serum albumin and dissolve it in 37.4 mL of the prepared PBS buffer solution 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 is 0.9:11, and the obtained mixture is stored at 3°C overnight; and the rest is the same as in Example 1. Finally, the artificial antigen II of lorcaserin is obtained.

[0099] Example 3

[0100] The difference between this example and Example 1 is that:

[0101] This example provides an artificial hapten I of lorcaserin, a preparation method thereof, and an artificial antigen II of lorcaserin and a preparation method thereof.

[0102] A preparation method of an artificial antigen of lorcaserin, wherein,

[0103] (1) The preparation process of the lorcaserin artificial hapten I: step (S.1) adjust the pH to 10 with ammonia water; step (S.3) dissolve 96 mg (0.491 mmol) of the colorless oily substance A obtained in step (S.2) in 13 mL of acetone, and add 305 mg (2.209 mmol) of potassium carbonate; then add 136 μL (1.227 mmol) of ethyl bromoacetate; step (S.4) prepare a 2 mol / L NaOH solution: dissolve 16 g of NaOH in 200 mL of deionized water to obtain a 2 mol / L NaOH solution; prepare a 1.5 mol / L hydrochloric acid solution: dilute 25.425 mL of 37% concentrated hydrochloric acid with deionized water to 200 mL to obtain a 1.5 mol / L hydrochloric acid solution. Then dissolve 137 mg (0.487 mmol) of the colorless oily substance B obtained in step (S.3) in a 50 mL single-necked flask with 3.73 mL of tetrahydrofuran and 3.73 mL of methanol, and add 14.95 mL of the prepared 2 mol / L NaOH solution; adjust the pH to 4 with the prepared 1.5 mol / L hydrochloric acid solution; and the rest is the same as in Example 1. Finally, the lorcaserin artificial hapten I is obtained.

[0104] (2) The preparation process of the lorcaserin artificial antigen II:

[0105] In step (a), dissolve 118 mg (0.466 mmol) of the lorcaserin artificial hapten I obtained in step (S.4) completely in 7.08 mL of N,N-dimethylformamide (DMF), and add 80.38 mg (0.699 mmol) of N-hydroxysuccinimide and 143.29 mg (0.699 mmol) of cyclohexylcarbodiimide, and stir the reaction at 30°C for 19 h; in step (b), weigh 348 mg of bovine serum albumin and dissolve it in 58 mL of the 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 is 1.1:9, and the obtained mixture is stored at 5°C overnight; and the rest is the same as in Example 1. Finally, the lorcaserin artificial antigen II is obtained.

[0106] Comparative Example 1

[0107] The difference between this comparative example and Example 1 is that:

[0108] This comparative example provides a lorcaserin artificial hapten I, a preparation method thereof, and a lorcaserin artificial antigen III and a preparation method thereof.

[0109] A preparation method of lorcaserin artificial antigen, wherein (1) the preparation process of lorcaserin artificial hapten I is the same as that in Example 1. (2) the preparation process of lorcaserin artificial antigen III: in step (b), the volume ratio of supernatant to bovine serum albumin solution is replaced by 1:5, instead of 1:10. The others are the same as those in Example 1. Finally, lorcaserin artificial antigen III is obtained. The synthetic route of lorcaserin artificial antigen III in this comparative example is the same as that in Example 1, and n is about 42.

[0110] Comparative Example 2

[0111] The difference between this comparative example and Example 1 is that:

[0112] This comparative example provides a lorcaserin artificial hapten I, a preparation method thereof, and a lorcaserin artificial antigen IV and a preparation method thereof.

[0113] A preparation method of lorcaserin artificial antigen, wherein (1) the preparation process of lorcaserin artificial hapten I is the same as that in Example 1. (2) the preparation process of lorcaserin artificial antigen IV: in step (b), bovine gamma globulin is used to replace bovine serum albumin (i.e. carrier protein) to couple with lorcaserin hapten I, and the same coupling steps as in Example 1 are adopted. The others are the same as those in Example 1. Finally, lorcaserin artificial antigen IV is obtained. The synthetic route of lorcaserin artificial antigen IV in this comparative example is shown as follows:

[0114]

[0115] wherein BGG is bovine gamma globulin, and n is about 18.

[0116] Comparative Example 3

[0117] The difference between this comparative example and Comparative Example 1 is that:

[0118] This comparative example provides a lorcaserin artificial hapten I, a preparation method thereof, and a lorcaserin artificial antigen V and a preparation method thereof.

[0119] A preparation method of lorcaserin artificial antigen, wherein (1) the preparation process of lorcaserin artificial hapten I is the same as that in Example 1. (2) the preparation process of lorcaserin artificial antigen V: in step (b), bovine gamma globulin is used to replace bovine serum albumin (i.e. carrier protein) to couple with lorcaserin hapten I, and the same coupling steps as in Comparative Example 1 are adopted. The others are the same as those in Comparative Example 1. Finally, lorcaserin artificial antigen V is obtained. The synthetic route of lorcaserin artificial antigen V in this comparative example is the same as that in Comparative Example 2, and n is about 15.

[0120] Comparative Example 4

[0121] The present comparative example provides a lorcaserin artificial hapten VI and a preparation method thereof, and a lorcaserin artificial antigen VII and a preparation method thereof.

[0122] A preparation method of a lorcaserin artificial antigen, comprising the following steps:

[0123] (1) Preparation of lorcaserin artificial hapten VI:

[0124] (S.1) A 10% sodium bicarbonate solution was prepared. A 1 mol / L NaOH solution was prepared: 8 g of NaOH was dissolved in 200 mL of deionized water to obtain a 1 mol / L NaOH solution. A 1 mol / L hydrochloric acid solution was prepared: 16.95 mL of 37% concentrated hydrochloric acid was diluted with deionized water to 200 mL to obtain a 1 mol / L hydrochloric acid solution. 100 mg (0.473 mmol) of 7-hydroxy lorcaserin was dissolved in 5 mL of deionized water in a 50 mL round-bottom flask, placed in an ice bath, and slowly adjusted to pH = 11 with the prepared 10% sodium bicarbonate solution to obtain a basic solution;

[0125] (S.2) 191 mg (0.567 mmol) of 9-fluorenylmethyl-N-succinimidyl carbonate was dissolved in 2.5 mL of 1,4-dioxane, and the basic solution obtained in step (S.1) was slowly added dropwise under ice bath, and then warmed to room temperature (25°C) and stirred for 18 h. TLC detection (developing agent: ethyl acetate) showed that the reaction was complete, and the reaction was stopped. 10 mL of deionized water was added, and the water phase was washed with 5 mL of ether for 3 times, and the water phase was collected. The water phase was adjusted to pH = 2 with the 1 mol / L hydrochloric acid solution prepared in step (S.1), and a large amount of white precipitate was generated. The precipitate was extracted with 10 mL of ethyl acetate for 3 times, and the ethyl acetate phase was collected. The organic phase was washed with 10 mL of deionized water and 10 mL of saturated brine, respectively, and then dried with anhydrous sodium sulfate, filtered, and dried to obtain 149 mg (0.344 mmol) of colorless oil A;

[0126] (S.3) Colorless oil A obtained in step (S.2) was dissolved in 50 mL of round-bottom flask with 14.9 mL of acetone, 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 for 18 h in the dark. TLC detection (developing agent: ethyl acetate: ammonia water = 6 mL: 3 drops) showed that the reaction was complete, and the product had a spot Rf = 0.5. Filtration, drying, and TLC separation and purification (developing agent: ethyl acetate: ammonia water = 6 mL: 3 drops, solvent and eluent: anhydrous ethanol) gave 159 mg (0.306 mmol) of colorless oil B;

[0127] (S.4) In a 50 mL round bottom flask, 144 mg (0.293 mmol) of the colorless oil B obtained in step (S.3) was 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 reaction was stirred at 25 °C for 18 h. The reaction product was centrifuged, and the supernatant was taken for use. Figure 3 Figure 3 The UV scan of the chlorocarpidil artificial hapten VI is shown in Figure 2.

[0128] (2) Preparation of chlorocarpidil artificial antigen VII:

[0129] (a) In a 50 mL single-necked round bottom flask, 144 mg (0.293 mmol) of the chlorocarpidil 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 reaction was stirred at 25 °C for 18 h. The reaction product was centrifuged, and the supernatant was taken for use.

[0130] (b) 14.5 g (40.503 mmol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (750 mmol) of sodium chloride, and 1.495 g (9.583 mmol) of sodium dihydrogen phosphate dihydrate were dissolved in deionized water and diluted to 5.0 L to obtain a 0.01 M PBS buffer with a pH of 7.4. 360 mg of bovine serum albumin was dissolved in 72 mL of the prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 5 mg / mL. Under rapid stirring, the supernatant obtained in step (a) was slowly added to the prepared bovine serum albumin solution, and the volume ratio of the supernatant to the bovine serum albumin solution was 1:10. The obtained mixture was stored at 4 °C overnight to obtain an artificial antigen mixture. The artificial antigen mixture was transferred to a dialysis bag and dialyzed against the prepared PBS buffer for 7 times. After dialysis, the supernatant was obtained by centrifugation to obtain the chlorocarpidil artificial antigen VII.

[0131] The synthetic route of the chlorocarpidil artificial antigen VII in the present comparative example is shown below: ​

[0132]

[0133] wherein Fmoc-Osu represents 9-fluorenylmethyl-N-succinimidyl carbonate, and n ~ 17.

[0134] Comparative Example 5

[0135] The difference between this comparative example and Comparative Example 4 is that:

[0136] This comparative example provides a chlorocarpaline artificial hapten VI and a preparation method thereof, and a chlorocarpaline artificial antigen VIII and a preparation method thereof.

[0137] A preparation method of a chlorocarpaline artificial antigen, wherein (1) the preparation process of chlorocarpaline artificial hapten VI is the same as that in Comparative Example 4. (2) The preparation process of chlorocarpaline artificial antigen VIII: in step (b), the volume ratio of supernatant to bovine serum albumin solution is replaced by 1:5 instead of 1:10. The others are the same as those in Comparative Example 4. Finally, chlorocarpaline artificial antigen VIII is obtained. The synthetic route of chlorocarpaline artificial antigen VIII in this comparative example is the same as that in Comparative Example 4, and n ~ 13.

[0138] Comparative Example 6

[0139] The difference between this comparative example and Comparative Example 4 is that:

[0140] This comparative example provides a chlorocarpaline artificial hapten VI and a preparation method thereof, and a chlorocarpaline artificial antigen IX and a preparation method thereof.

[0141] A preparation method of a chlorocarpaline artificial antigen, wherein (1) the preparation process of chlorocarpaline artificial hapten VI is the same as that in Comparative Example 4. (2) The preparation process of chlorocarpaline artificial antigen IX: in step (b), bovine gamma globulin is used to replace bovine serum albumin (i.e. carrier protein) to couple with chlorocarpaline hapten VI, and the same coupling steps as in Comparative Example 4 are used. The others are the same as those in Comparative Example 4. Finally, chlorocarpaline artificial antigen IX is obtained.

[0142] The synthetic route of chlorocarpaline artificial antigen IX in this comparative example is as follows:

[0143]

[0144] n ~ 7.

[0145] Comparative Example 7

[0146] The difference between this comparative example and Comparative Example 4 is that:

[0147] The present comparative example provides a chlorocarpalin artificial hapten VI and a preparation method thereof, and a chlorocarpalin artificial antigen X and a preparation method thereof.

[0148] A preparation method of a chlorocarpalin artificial antigen, wherein (1) the preparation process of chlorocarpalin artificial hapten VI is the same as that in Comparative Example 4. (2) The preparation process of chlorocarpalin artificial antigen IX: in step (b), bovine serum albumin (i.e. carrier protein) is replaced with bovine gamma globulin, and is coupled with chlorocarpalin hapten VI, using the same coupling steps as in Comparative Example 5, and the others are the same as in Comparative Example 4. Finally, chlorocarpalin artificial antigen X is obtained. The synthetic route of chlorocarpalin artificial antigen X in the present comparative example is the same as that in Comparative Example 6, and n≈4 at this time.

[0149]

Performance determination of chlorocarpalin artificial antigen

[0150] (1) Identification of chlorocarpalin artificial antigen:

[0151] Molar absorption coefficient ε: chlorocarpalin artificial hapten I and chlorocarpalin artificial hapten VI were prepared according to the methods in Example 1 and Comparative Example 4, respectively. Then, chlorocarpalin artificial hapten solutions with concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL were prepared using buffer, respectively. Through ultraviolet scanning diagram, it can be known that the maximum absorption wavelength of chlorocarpalin artificial hapten I is 248 nm, and the maximum absorption wavelength of artificial hapten VI is 266 nm. The absorbance values of the corresponding antigens were measured at 248 nm and 266 nm, respectively, and parallel samples were prepared for each concentration. The calculation formula of molar absorption coefficient (i.e. molar absorption coefficient) is: ε = absorbance / molar concentration. The calculation data results are shown in Table 1 below.

[0152] Table 1: Calculation table of molar absorption coefficient of chlorocarpalin artificial hapten I and VI

[0153]

[0154] Determination of the concentration of the conjugate protein: The artificial antigen of lorcaserin was prepared according to the method of Example 1 and Comparative Examples 1-7. 1 mL of bovine serum albumin solution with concentrations of 0, 10, 20, 30, 40, 60, 80, 100, and 120 μg / mL was prepared with PBS buffer, 3 mL of Coomassie brilliant blue staining solution was added, mixed immediately, and heated in a water bath at 30°C for 5 min. Parallel samples were prepared for each concentration, and the absorbance was measured at 655 nm. The relationship between the protein concentration and the absorbance was plotted. The artificial antigen solution (prepared with PBS buffer) was diluted at a certain ratio, and the absorbance of the artificial antigen was measured at 655 nm. The corresponding protein concentration of the artificial antigen solution was read from the curve, and the data results are shown in Table 2.

[0155] Determination of the coupling ratio: when the hapten of the conjugate is the artificial hapten VI of lorcaserin, 100 μg / mL of bovine serum albumin PBS solution and 100 μg / mL of bovine gamma globulin PBS solution were prepared. The conjugate (i.e., the artificial antigen of lorcaserin) was diluted with PBS to 100 μg / mL, and the absorbance A1 was measured at 266 nm. The absorbance A2 was measured with PBS as a blank (selected according to the protein coupled with the artificial antigen of lorcaserin). 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 calculation data results are shown in Table 2.

[0156] When bovine gamma globulin is used as the carrier, the calculation formula of the coupling ratio γ is: γ = [(A1-A2) / ε] / (100×10 -3 / 43000), where 43000 is the molecular weight of bovine gamma globulin, and 100×10 -3 is the concentration of bovine gamma globulin (g / L). The calculation data results are shown in Table 2.

[0157] Table 2: Calculation data results of the coupling ratio of each artificial antigen of lorcaserin and the protein concentration of the conjugate

[0158]

[0159] As shown in Tables 1-2, the structure of the artificial hapten, the volume ratio between the artificial hapten and the carrier protein solution when they are coupled, and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten is cross-linked with the carrier protein.

[0160] (2) Animal immunization

[0161] Chlorocarpaline artificial antigens were prepared according to the method of Example 1 and Comparative Examples 1-7, respectively. The prepared chlorocarpaline artificial antigens were used to immunize New Zealand white rabbits, and the obtained immune sera were detected by ELISA. The detection results are shown in Table 3 below.

[0162] Table 3: Detection results of the titers of the immune sera

[0163] Number Clocapramine 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 / .

[0164] As shown in Table 3, compared with Example 1, the titers of the immune sera obtained by immunizing animals with the chlorocarpaline artificial antigens prepared by the methods of the comparative examples are all low, and cannot be used in immunoassay. The titer of the immune serum obtained by immunizing animals with the chlorocarpaline artificial antigen II prepared by the method of Example 1 is 1:256000, and can be completely used in immunoassay, and can provide a more convenient, rapid and accurate way for the detection of chlorocarpaline.

[0165] The above only describes the preferred embodiments and principles of the present application in detail. For those skilled in the art, the specific embodiments can be changed according to the idea provided by the present application, and these changes should be considered as the protection scope of the present application.

Claims

1. A chlorocarpaline artificial hapten, characterized in that, The molecular structure is shown as formula (I).

2. A method for preparing a lorcaserin artificial hapten according to claim 1, characterized in that, The method comprises the following steps: (S.1) dissolving the lorcaserin hydrochloride in deionized water, adjusting the pH to alkaline with ammonia water 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 colorless oil A; (S.3) dissolving the colorless oil A obtained in step (S.2) in a solvent, adding potassium carbonate, then adding ethyl bromoacetate, uniformly mixing and heating and stirring to react, filtering after the reaction is completed, and drying to obtain colorless oil B; (S.4) respectively preparing a sodium hydroxide solution and a hydrochloric acid solution, then adding tetrahydrofuran, methanol, and the prepared sodium hydroxide solution to the colorless oil B obtained in step (S.3), heating and stirring to react until the reaction is completed, adjusting the pH to be acidic with the prepared hydrochloric acid solution, drying, extracting with anhydrous ethanol, filtering, drying, and separating to obtain the lorcaserin artificial hapten I.

3. A process for the preparation of a lorcaserin artificial hapten according to claim 2, characterized by, The pH of the alkaline solution in step (S.1) is 8-10; In step (S.3), the molar ratio of the colorless oil A 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; and the feed 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, In step (S.4), the feed liquid ratio of the colorless oil B, tetrahydrofuran, methanol, and the sodium hydroxide solution 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, The molecular structure is shown as formula (II): BSA is bovine serum albumin, and n is 55-60.

6. The method for preparing a lorcaserin artificial antigen according to claim 5, wherein the lorcaserin is a compound represented by the following formula (I) : ###0001### (I). The method comprises the following steps: coupling the lorcaserin artificial hapten I according to claim 1 with bovine serum albumin by using an N-hydroxysuccinimide active ester method, and purifying to obtain the lorcaserin artificial antigen II.

7. The method for preparing a lorcaserin artificial antigen according to claim 6, characterized in that, The method comprises the following steps: (a) mixing the lorcaserin artificial hapten I, N-hydroxysuccinimide, and cyclohexylcarbonyldiimide in N,N-dimethylformamide, heating and stirring to react until the reaction is completed, centrifuging, and taking the supernatant; (b) adding the supernatant obtained in step (a) to a bovine serum albumin solution, uniformly mixing and standing overnight, dialyzing, centrifuging to take the supernatant, and obtaining the lorcaserin artificial antigen II.

8. The method according to claim 7, wherein The molar ratio of the chlorocarpaline artificial hapten, N-hydroxysuccinimide, and cyclohexyl carbodiimide added in step (a) is 1: (1.35-1.5): (1.35-1.5); the ratio of chlorocarpaline artificial hapten to 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℃, and the reaction time is 18-20 h; In step (b), the volume ratio of the supernatant 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 the overnight standing is 3-5℃.

9. Use of the chlorocarpaline artificial antigen of claim 5 in the preparation of chlorocarpaline antibodies.

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