Preparation and application of N-acetyl-5-hydroxytryptamine derivatives, immunogens, antibodies and detection reagents

Abstract

5-Hydroxytryptamine (5-HT) was first discovered in serum and is also known as serotonin. Due to its small molecular weight and poor immunogenicity, the antibodies prepared with 5-HT do not meet the specificity and affinity requirements of immunoassay reagents. This invention innovatively uses N-acetyl-5-hydroxytryptamine, with a larger molecular weight, for hapten design. By coupling it with a specific macromolecular carrier, the prepared intact antigen possesses excellent specificity and strong affinity. This invention mainly relates to the design and synthesis of N-acetyl-5-hydroxytryptamine haptens, the preparation of intact N-acetyl-5-hydroxytryptamine antigens and anti-N-acetyl-5-hydroxytryptamine antibodies, as well as methods for determining 5-hydroxytryptamine concentration, and the composition and components of the detection reagents, belonging to the field of biomedical detection technology. The N-acetyl serotonin derivative of this invention has the structure shown in formula (Ⅰ): where R is —(CH2). n —COOH, where n is an integer between 1 and 10.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to an N-acetyl-5-hydroxytryptamine derivative, an N-acetyl-5-hydroxytryptamine immunogen and its specific antibody, and a 5-hydroxytryptamine detection kit. Background Technology

[0002] N-Acetyl-5-hydroxytryptamine, its structural formula is shown in formula (Ⅲ):

[0003]

[0004] Serotonin, an intermediate product of tryptophan metabolism, is mainly found in enterochromaffin cells, serotonin-activated neurons, and platelets. It has been identified as a neurotransmitter in the central nervous system. Almost all circulating serotonin is concentrated in platelets. Changes in circulating serotonin concentration can reflect several pathological changes in the human body, including chronic tension headaches, schizophrenia, hypertension, chorea, Duchenne muscular dystrophy, and early-stage acute appendicitis. Furthermore, the detection of serum serotonin is of significant clinical importance in the detection of carcinoid syndromes.

[0005] Currently, the main methods for detecting serotonin include fluorescence analysis, enzyme-linked immunosorbent assay (ELISA), gas chromatography, and high-performance liquid chromatography (HPLC). These methods are cumbersome, time-consuming, and labor-intensive, and cannot simultaneously guarantee accuracy and timeliness in clinical testing. This invention addresses these pain points in clinical serotonin testing by starting with antigen design and antibody preparation, and employing an advanced, fully automated chemiluminescence detection platform. It has broad application prospects. This test reagent enables high-throughput, automated, and multi-sample testing, and boasts outstanding advantages in terms of high sensitivity, specificity, and stability. Summary of the Invention

[0006] The purpose of this invention is to provide an N-acetyl-5-hydroxytryptamine derivative, an N-acetyl-5-hydroxytryptamine immunogen and its specific antibody and a 5-hydroxytryptamine detection kit, so as to improve the shortcomings of existing 5-hydroxytryptamine determination methods, such as poor specificity, low sensitivity and inability to perform automated analysis.

[0007] According to one aspect of the present invention, an N-acetyl-5-hydroxytryptamine derivative is provided, having the structural formula shown in formula (I):

[0008]

[0009] Wherein, R is a linking group -(CH2)n-COOH, and n is an integer between 1 and 10. The N-acetyl-5-hydroxytryptamine derivative of the present invention possesses the basic structure for preparing an immunogenic N-acetyl-5-hydroxytryptamine immunogen, providing a structural basis for the preparation of 5-hydroxytryptamine detection reagents.

[0010] According to another aspect of the present invention, an N-acetyl-5-hydroxytryptamine immunogen is also provided, which has the structural formula shown in formula (II):

[0011]

[0012] Formula (II)

[0013] Wherein R is a linking group -(CH2)n-COOH, n is an integer between 1 and 10, and the carrier is an immunogenic protein or polypeptide. The N-acetyl-5-hydroxytryptamine immunogen of this invention exhibits high immunogenicity, can stimulate an immune response in animals, and produces high-titer anti-N-acetyl-5-hydroxytryptamine specific antibodies. It also possesses strong antibody affinity, making it suitable for preparing highly sensitive and specific 5-hydroxytryptamine competitive assay reagents.

[0014] When n=1, R in the above N-acetyl-5-hydroxytryptamine immunogen is -CH2-COOH. A protein carrier is preferred as a carrier for the above N-acetyl-5-hydroxytryptamine immunogen; however, other types of immunogenic substances with sufficiently large molecular weights and a sufficient number of active groups can also be used as carriers. The most commonly used immunogenic carriers include serum proteins, keyhole hemocyanin (KLH), thyroglobulin, and polylysine. The carrier in this invention is preferably keyhole hemocyanin.

[0015] According to another aspect of the present invention, an anti-N-acetyl-5-hydroxytryptamine specific antibody is also provided, produced by immunizing animals with an immunogen, wherein the antibody is produced by immunizing animals with any of the aforementioned N-acetyl-5-hydroxytryptamine immunogens. The term "antibody" as used in this invention refers not only to complete protein molecules but also to polypeptide fragment antibodies or polypeptide fragment antibody derivatives that retain the specific binding ability of complete antibodies. The antibody of the present invention can be a polyclonal antibody, a monoclonal antibody, or a recombinant antibody, preferably a monoclonal antibody.

[0016] The antibodies of this invention can be prepared using existing technologies. A typical method for obtaining polyclonal antibodies is to use a single immunogen, with or without adjuvant, to immunize an animal at one or more sites. Host animals include rabbits, goats, mice, sheep, guinea pigs, horses, alpacas, or camels. Continuous immunization is performed 5-7 times until the antibody titer reaches its maximum. Blood is collected from the animal at regular intervals to obtain appropriate amounts of specific antiserum. Monoclonal antibodies can be prepared using hybridoma cell technology. Recombinant antibodies can be prepared using gene engineering expression technology.

[0017] According to another aspect of the present invention, a method for preparing an N-acetyl-5-hydroxytryptamine immunogen is also provided. This method includes the steps of preparing the aforementioned N-acetyl-5-hydroxytryptamine derivative and linking the aforementioned N-acetyl-5-hydroxytryptamine derivative to a carrier to obtain the N-acetyl-5-hydroxytryptamine immunogen. The N-acetyl-5-hydroxytryptamine derivative prepared by the above method can be linked to an immunogenic protein or polypeptide to obtain the highly immunogenic N-acetyl-5-hydroxytryptamine immunogen of the present invention, and the preparation method is simple to operate.

[0018] In the above-described method for preparing the N-acetyl-5-hydroxytryptamine immunogen of the present invention, a method for preparing the above-described N-acetyl-5-hydroxytryptamine derivative is also provided, the method comprising the following:

[0019] S1. N-acetyl-5-hydroxytryptamine was subjected to a substitution reaction with halo-(CH2)n-COO-tert-butyl ester in an N,H-dimethylformamide environment to obtain 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0020] S2. Dissolve 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain an N-acetyl-5-hydroxytryptamine derivative.

[0021] This preparation method involves substituting the hydrogen at the 5-position of N-acetyl-5-hydroxytryptamine with halogenated -(CH2)n-COO-tert-butyl ester to generate 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine. Then, under acidic conditions, the 2-(CH2)n-COO-tert-butyl ester bond on the 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine is broken to form an N-acetyl-5-hydroxytryptamine derivative with -(CH2)n-COOH at the 5-position. This preparation method is simple to operate, has mild reaction conditions, high stability, and good reproducibility.

[0022] In step S1 of the preparation of the above-mentioned N-acetyl-5-hydroxytryptamine derivative, the following steps are also included:

[0023] A1. Dissolve N-acetyl-5-hydroxytryptamine and halo-(CH2)n-COO-tert-butyl ester in N,N-dimethylformamide (DMF) to obtain a mixed solution;

[0024] A2. Add ethyl acrylate to the mixed solution to obtain a solid-liquid mixture;

[0025] A3. The solid-liquid mixture was filtered to obtain 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine. N-acetyl-5-hydroxytryptamine and halo-(CH2)n-COO-tert-butyl ester are well soluble in DMF organic solvent. Ethyl acrylate was added to precipitate 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine, and the reactants were obtained by filtration.

[0026] In step A1 above, after obtaining the mixed solution, the process further includes heating the mixed solution to a constant temperature. Preferably, the temperature for heating to a constant temperature is 70–100°C, and the holding time is greater than or equal to 32 hours. Heating the mixed solution to a constant temperature and maintaining it for more than 32 hours ensures complete dissolution and reaction.

[0027] In this invention, in step A2 above, the reaction mixture is subjected to low-temperature stirring and solidification at 0-8°C to increase the yield.

[0028] In the preparation method of the N-acetyl-5-hydroxytryptamine derivative of the present invention, there are no special requirements for the specific operation of filtering the solid-liquid mixture to obtain 2-(CH2)-COO-tert-butyl N-acetyl-5-hydroxytryptamine, as long as the desired target product can be separated from the solid-liquid mixture. In the present invention, step A3 includes filtering the solid-liquid mixture to obtain a solid substance; washing and purifying the solid substance with acetone to obtain a purified product; and vacuum drying the purified product to obtain 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine. After the steps of filtration, washing and purification, and vacuum drying, the obtained 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine has relatively high purity and yield.

[0029] In the preparation method of the N-acetyl-5-hydroxytryptamine derivative of the present invention, the specific operation of the step of dissolving 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain the N-acetyl-5-hydroxytryptamine derivative can be appropriately adjusted according to the different 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine and acid solutions. In the present invention, step S2 preferably includes: dissolving 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain a reaction solution; stirring the reaction solution at 40-60°C for 2.5-6.5 h to obtain a stirred solution; drying the stirred solution to obtain a dried product; and washing and purifying the dried product with acetone to obtain the N-acetyl-5-hydroxytryptamine derivative. Stirring at a high temperature of around 60°C can promote the formation of the target N-acetyl-5-hydroxytryptamine derivative. The dried product containing the target N-acetyl-5-hydroxytryptamine derivative is obtained by evaporation and drying. After washing with acetone, the organic residues on the dried product are removed, and the target N-acetyl-5-hydroxytryptamine derivative with higher purity is obtained.

[0030] In the preparation method of the above-mentioned N-acetyl-5-hydroxytryptamine derivative of the present invention, when n=1, the preparation steps of the above-mentioned N-acetyl-5-hydroxytryptamine derivative are as follows:

[0031]

[0032] When n=1, the reaction steps are the same as above, except that tert-butyl bromoacetate is used as the raw material. Therefore, the linking group R of the final product N-acetyl-5-hydroxytryptamine derivative is -CH2-COO-.

[0033] In the method for preparing the N-acetyl-5-hydroxytryptamine immunogen of the present invention, the linking step between the carrier and the N-acetyl-5-hydroxytryptamine derivative can be reasonably modified in practice depending on the carrier. In the present invention, the linking step includes:

[0034] S1. Prepare a carrier solution and an N-acetyl-5-hydroxytryptamine derivative solution; wherein the mass ratio of the carrier to the N-acetyl-5-hydroxytryptamine derivative is 1 to 8:1; preferably, the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine;

[0035] S2. The activated N-acetyl-5-hydroxytryptamine derivative solution is added dropwise to the carrier solution to obtain crude N-acetyl-5-hydroxytryptamine immunogen;

[0036] S3. Stir the dropwise mixture overnight at 2-10°C or react at room temperature for 2 hours to obtain the crude N-acetyl-5-hydroxytryptamine immunogen.

[0037] S4. The crude N-acetyl-5-hydroxytryptamine immunogen is purified to obtain N-acetyl-5-hydroxytryptamine immunogen. The preparation steps of this invention can obtain the target product through simple activation, dropwise addition, and purification steps. The preparation method is simple, has high process stability, and good reproducibility.

[0038] In the above-described method for preparing the N-acetyl-5-hydroxytryptamine immunogen of the present invention, the actual operation of the steps for preparing the carrier solution and the N-acetyl-5-hydroxytryptamine derivative solution involves rationally selecting suitable solvent concentrations and pH values ​​according to the different types of carriers. In step S1 of the present invention, the step of preparing the carrier solution involves dissolving the carrier in 0.05–0.20 M phosphate buffer solution with a pH of 8.0–9.5 to obtain the carrier solution; the step of preparing the N-acetyl-5-hydroxytryptamine derivative solution involves placing the N-acetyl-5-hydroxytryptamine derivative, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide, and N-hydroxythiosuccinimide in N,N-dimethylformamide, methanol, and 5–20 mM potassium phosphate buffer solution with a pH of 4.0–6.0 and stirring at room temperature to obtain an activated N-acetyl-5-hydroxytryptamine derivative solution. Dissolving the carrier in a phosphate buffer solution with a concentration of 0.10–0.25 M and a pH of 8.0–9.5 allows for sufficient reaction and binding of the amino groups on the carrier and the carboxyl groups of the activated N-acetyl-5-hydroxytryptamine derivative in a slightly alkaline environment. Using a phosphate buffer solution with a concentration of 5–20 mM and a pH range of 4.0–6.0 allows for sufficient activation of the carboxyl groups of the N-acetyl-5-hydroxytryptamine derivative in a slightly acidic solution.

[0039] In the above-described method for preparing N-acetyl-5-hydroxytryptamine immunogen of the present invention, in the step of obtaining crude N-acetyl-5-hydroxytryptamine immunogen by dropwise addition, in order to further increase the content of N-acetyl-5-hydroxytryptamine immunogen in the crude N-acetyl-5-hydroxytryptamine immunogen, in steps S2 and S3 of the present invention, the dropwise addition step allows the N-acetyl-5-hydroxytryptamine derivative to react more fully with the carrier; stirring overnight at 2-10°C further promotes the generation of N-acetyl-5-hydroxytryptamine immunogen, thereby increasing the content of N-acetyl-5-hydroxytryptamine immunogen in the crude product.

[0040] In the above-described method for preparing N-acetyl-5-hydroxytryptamine immunogen of the present invention, any operation capable of purifying N-acetyl-5-hydroxytryptamine immunogen from crude N-acetyl-5-hydroxytryptamine immunogen is applicable to the present invention. Preferably, in step S4 above, the crude N-acetyl-5-hydroxytryptamine immunogen is purified by dialysis to obtain N-acetyl-5-hydroxytryptamine immunogen, as dialysis is a simple method and has good purification effect.

[0041] According to another aspect of the present invention, an N-acetyl-5-hydroxytryptamine detection reagent is also provided, comprising an anti-N-acetyl-5-hydroxytryptamine specific antibody, an N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate, and an enzyme substrate, wherein the anti-N-acetyl-5-hydroxytryptamine specific antibody is any one of the aforementioned anti-N-acetyl-5-hydroxytryptamine specific antibodies; the N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate contains the aforementioned N-acetyl-5-hydroxytryptamine derivative, and the N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate is formed by conjugation of an enzyme and a hapten, wherein the hapten is the aforementioned N-acetyl-5-hydroxytryptamine derivative.

[0042] The N-acetyl-5-hydroxytryptamine detection reagent of the present invention exhibits significantly higher detection sensitivity than corresponding products in the prior art due to the high specificity of the anti-N-acetyl-5-hydroxytryptamine specific antibody and its strong binding affinity to N-acetyl-5-hydroxytryptamine. Preferably, the enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; the substrate of the enzyme is AMPPD or APS-5. The detection reagent using an alkaline phosphatase-hapten enzyme-labeled conjugate and AMPPD or APS-5 as the enzyme substrate can conveniently and accurately determine the N-acetyl-5-hydroxytryptamine content in a sample, making it suitable for high-throughput automated detection.

[0043] According to another aspect of the present invention, a 5-hydroxytryptamine (5-HT) detection kit is also provided, comprising the above-mentioned anti-N-acetyl-5-hydroxytryptamine specific antibody and an indicator reagent for detecting the anti-N-acetyl-5-hydroxytryptamine specific antibody and the N-acetyl-5-hydroxytryptamine complex. The indicator reagent is selected from enzyme reagents, radioisotope reagents, fluorescent reagents, and luminescent reagents. Preferably, the indicator reagent consists of an N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate and an enzyme substrate, wherein the N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate can be coupled with the N-acetyl-5-hydroxytryptamine derivative of the present invention, enabling convenient and accurate determination of the N-acetyl-5-hydroxytryptamine content in a sample, suitable for high-throughput automated detection.

[0044] The present invention utilizes an N-acetyl-5-hydroxytryptamine derivative obtained by substituting a specific hydrogen atom at a specific site with a specific R1 group. This derivative, linked to a specific carrier, forms an N-acetyl-5-hydroxytryptamine immunogen with high immunogenicity. The antibodies induced in immunized animals exhibit high specificity and a strong specific binding affinity to N-acetyl-5-hydroxytryptamine. High-throughput and rapid detection of N-acetyl-5-hydroxytryptamine can be achieved using alkaline phosphatase chemiluminescence immunoassay technology on a fully automated chemiluminescence immunoassay analyzer. This method offers advantages such as ease of operation, high sensitivity, strong specificity, and accurate results, while also effectively reducing the cost of N-acetyl-5-hydroxytryptamine detection, thus facilitating its widespread clinical application. Attached Figure Description

[0045] Appendix Figure 1: Standard curve for N-acetyl-5-hydroxytryptamine ELISA detection.

[0046] Appendix Figure 2 Chemiluminescence calibration curve of N-acetyl-5-hydroxytryptamine alkaline phosphatase.

[0047] Appendix Figure 3 The results of comparing the N-acetyl-5-hydroxytryptamine chemiluminescent detection reagent of the present invention with the ELISA detection reagent of a comparative manufacturer. Detailed Implementation

[0048] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0049] Example 1: Synthesis and structural confirmation of N-acetyl-5-hydroxytryptamine derivatives

[0050] The chemical structure of the N-acetyl-5-hydroxytryptamine derivative used in the following examples is shown in formula (Ⅳ):

[0051]

[0052] The specific synthetic steps of the N-acetyl-5-hydroxytryptamine derivative shown in formula (Ⅳ) are as follows:

[0053] Synthesis of S1 and Compound 2

[0054]

[0055] The specific synthesis steps of compound 2 are as follows:

[0056]

[0057] 15.0 g of compound 1 and 26 g of tert-butyl bromoacetate were weighed and dissolved together in 150 mL of N,N-dimethylformamide (DMF). The solution was then heated to 95 °C and left overnight. 500 mL of ethyl acrylate (EA) was added to this solution, and the reaction mixture was stirred at 0 °C for 25 min. The solid precipitate in the solution was filtered off, washed with acetone, and dried under vacuum to obtain 11.2 g of compound 2 as a brown solid, with a yield of 30%.

[0058] Synthesis of S2, N-acetyl-5-hydroxytryptamine derivatives

[0059]

[0060] A1. Weigh 3.00 g of compound 2 and dissolve it in 30 mL of HCl (1.5 M). Stir the solution at 60 °C for 3 hours. Evaporate the reaction mixture to dryness, and wash the residue with acetone to finally obtain 2.10 g of a reddish-brown solid N-acetyl-5-hydroxytryptamine derivative, with a yield of 92%.

[0061] A2. The above compound was subjected to nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as an internal standard. The result was characterized as 2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0062] Example 2: Synthesis and preparation of derivatives when n=2

[0063] Synthesis of S1,2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine

[0064] A1. Weigh 18.0 g of compound 1 and 27.8 g of tert-butyl bromopropionate, and dissolve them together in 150 mL of N,N-dimethylformamide (DMF). Heat the solution to 95 °C overnight. Add 550 mL of ethyl acrylate (EA) to this solution, and stir the reaction mixture at 4 °C for 25 min. Filter the solid precipitate from the solution, wash with acetone, and dry under vacuum to obtain 7.70 g of a brown solid intermediate, with a yield of 26%.

[0065] A2. The above compound was subjected to nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as an internal standard. The result was characterized as 2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0066] Synthesis of S2, N-acetyl-5-hydroxytryptamine derivatives

[0067] A1. Weigh 3.00 g of the above-mentioned 2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine, dissolve it in 30 mL of HCl (1.5 M), and stir the solution at 60 °C for 2 hours. Evaporate the reaction mixture to dryness, and wash the residue with acetone to finally obtain 2.1 g of reddish-brown solid N-acetyl-5-hydroxytryptamine derivative, with a yield of 92.1%.

[0068] A2. Structural identification of the purified product obtained above.

[0069] a. The above compound was subjected to nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as an internal standard. The result was characterized as 2-(CH2)2-COO-N-acetyl-5-hydroxytryptamine.

[0070] b. The obtained derivative was analyzed and identified by chromatography / mass spectrometry (LC / MS), and the final compound was determined to be 2-(CH2)2-COO-N-acetyl-5-hydroxytryptamine.

[0071] Example 3: Synthesis and preparation of N-acetyl-5-hydroxytryptamine derivatives when n=3

[0072] Synthesis of S1,2-(CH2)3-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine

[0073] A1. Weigh 18.0 g of compound 1 and 29.5 g of tert-butyl bromobutyrate, and dissolve them together in 180 mL of N,N-dimethylformamide (DMF). Heat the solution to 95 °C overnight. Add 525 mL of ethyl acrylate (EA) to this solution, and stir the reaction mixture at 5 °C for 30 min. Filter the solid precipitate from the solution, wash with acetone, and dry under vacuum to obtain 7.2 g of white solid compound 2, with a yield of 22%.

[0074] A2. The above compound was subjected to nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as an internal standard. The results showed that the compound was 2-(CH2)3-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0075] Synthesis of S2, N-acetyl-5-hydroxytryptamine derivatives

[0076] A1. Weigh 3.00 g of compound 2 and dissolve it in 30 mL of HCl (1.5 M). Stir the solution at 55 °C for 4.5 hours. Evaporate the reaction mixture to dryness, and wash the residue with acetone to finally obtain 2.1 g of a brown solid N-acetyl-5-hydroxytryptamine derivative, with a yield of 94%.

[0077] A2. Structural identification of the purified product obtained above.

[0078] a. The above compound was subjected to nuclear magnetic resonance spectroscopy using Varian III plus 300MHz, with TMS as an internal standard. The result was characterized as 2-(CH2)3-COO-N-acetyl-5-hydroxytryptamine.

[0079] b. The obtained derivative was analyzed and identified by chromatography / mass spectrometry (LC / MS), and the final compound was determined to be 2-(CH2)3-COO-N-acetyl-5-hydroxytryptamine.

[0080] In the above embodiments, when n = 1, 2, and 3, the preparation of N-acetyl-5-hydroxytryptamine derivatives used tert-butyl bromoacetate, tert-butyl bromopropionate, and tert-butyl bromobutyrate as synthetic raw materials during the synthesis of intermediate compounds, respectively. Therefore, the linking groups R1 of the final N-acetyl-5-hydroxytryptamine derivatives obtained are -CH2-COO-, -(CH2)2-COO-, and -(CH2)3-COO-, respectively. When n is any other integer from 1 to 10, the synthesis method is completely consistent when other tert-butyl bromoorganic acids similar to tert-butyl bromoacetate are used in the experiment, except for the different values ​​of n.

[0081] Example 4: Synthesis of BSA-N-acetyl-5-hydroxytryptamine derivative immunogen

[0082] The BSA-N-acetyl-5-hydroxytryptamine immunogen is formed by linking bovine serum albumin (BSA) with the -(CH2)n-COO- group of the N-acetyl-5-hydroxytryptamine derivative shown in formula (I). In this embodiment, the synthesis method of this immunogen is described in detail with n=1 as an example. The specific steps are as follows:

[0083] S1. Dissolve bovine serum albumin (20 mg) in 5 ml of 0.2 M phosphate buffer, pH 8.5;

[0084] S2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized N-acetyl-5-hydroxytryptamine derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM, pH 5.0 phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.5 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 min.

[0085] S3. Add the activated solution dropwise to the BSA solution and stir overnight at 2-10℃ to obtain the crude antigen. Purify the synthesized antigen by dialysis to obtain the 2-acetic acid N-acetyl-5-hydroxytryptamine immunogen.

[0086] Example 5: Synthesis of KLH-N-acetyl-5-hydroxytryptamine derivative immunogen

[0087] The KLH-acetyl-5-hydroxytryptamine immunogen is formed by linking hemocyanin (KLH) with the -(CH2)n-COO- group of the N-acetyl-5-hydroxytryptamine derivative shown in formula (Ⅰ). In this embodiment, the synthesis method of this immunogen is described in detail with n=2 as an example. The specific steps are as follows:

[0088] S1. Dissolve hemocyanin (20 mg) in 5 ml of 0.18 M, pH 8.5 phosphate buffer;

[0089] S2. Add the following chemicals to a small beaker and stir to dissolve: 20 mg of the synthesized N-acetyl-5-hydroxytryptamine derivative, 0.3 ml of D,D-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 10 mM, pH 5.0 phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and dissolve these chemicals at room temperature for 10 min.

[0090] S3. Add the activated solution dropwise to the KLH solution and stir overnight at 2-10℃ to obtain the crude N-acetyl-5-hydroxytryptamine immunogen; purify the synthesized antigen by dialysis to obtain 2-propionic acid N-acetyl-5-hydroxytryptamine immunogen.

[0091] Example 6: Synthesis of immunogen from thyroglobulin N-acetyl-5-hydroxytryptamine derivative

[0092] The thyroglobulin N-acetyl-5-hydroxytryptamine immunogen is formed by linking thyroglobulin with the -(CH2)n-COO- group of the N-acetyl-5-hydroxytryptamine derivative shown in formula (Ⅰ). In this embodiment, the synthesis method of this immunogen is described in detail with n=3 as an example. The specific steps are as follows:

[0093] S1. Dissolve thyroglobulin (20 mg) in 5 ml of 0.20 M, pH 9.0 phosphate buffer;

[0094] S2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized N-acetyl-5-hydroxytryptamine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and react these chemicals at room temperature for 30 min.

[0095] S3. Add the activated solution dropwise to the thyroglobulin solution and stir overnight at 2-10℃ to obtain the complete antigen; purify the synthesized antigen by dialysis to obtain 2-butyric acid N-acetyl-5-hydroxytryptamine immunogen.

[0096] Example 7: Synthesis of poly-L-lysine-N-acetyl-5-hydroxytryptamine derivative immunogen

[0097] The poly-L-lysine-N-acetyl-5-hydroxytryptamine immunogen is formed by linking poly-L-lysine with the -(CH2)n-COO- group of the N-acetyl-5-hydroxytryptamine derivative shown in formula (Ⅰ). In this embodiment, the synthesis method of the immunogen is described in detail with n=3 as an example. The specific steps are as follows:

[0098] S1. Dissolve poly-L-lysine (20 mg) in 5 ml of 0.20 M phosphate buffer solution at pH 9.0;

[0099] S2. Add the following chemicals to a small beaker and stir to dissolve: 10 mg of the synthesized N-acetyl-5-hydroxytryptamine derivative, 0.3 ml of N,N-dimethylformamide (DMF), 0.3 ml of methanol, 1.0 ml of 20 mM, pH 5.0 potassium phosphate buffer, 20 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and 2.0 mg of N-hydroxythiosuccinimide (Sulfo-NHS). Stir and react these chemicals at room temperature for 30 min.

[0100] S3. Add the activated solution dropwise to the poly-L-lysine solution and stir overnight at 2-10℃ to obtain the complete antigen. Centrifuge the synthesized antigen and collect the supernatant. Purify the supernatant by dialysis to obtain the 2-butyric acid N-acetyl-5-hydroxytryptamine immunogen.

[0101] Similarly, when n takes other integer values ​​from 1 to 10, the N-acetyl-5-hydroxytryptamine immunogen shown in formula (II) can be prepared using the same method, and the experimental results show no significant difference. N-acetyl-5-hydroxytryptamine immunogens prepared using N-acetyl-5-hydroxytryptamine derivatives with different n values ​​all exhibit strong immunogenicity, and the corresponding specific antibodies prepared all have excellent performance. Of course, the carrier is still an immunogenic protein, which can be serum protein, keyhole hemocyanin (KLH), thyroglobulin, and polylysine. Preferably, the carrier is keyhole hemocyanin.

[0102] Example 8: Preparation of anti-N-acetyl-5-hydroxytryptamine specific monoclonal antibody

[0103] This embodiment presents a method for preparing N-acetyl-5-hydroxytryptamine monoclonal antibody, which includes the following steps:

[0104] S1, Animal Immunization

[0105] The artificial antigen prepared in Example 5 was used to immunize two approximately 8-week-old female Balb / C mice via subcutaneous injection at multiple points on the neck and back. For the initial immunization, an immunogen emulsified with Freund's complete adjuvant was used. Equal volumes of immunogen and Freund's complete adjuvant were mixed and emulsified, with a single mouse immunogen dose of 0.1 mg. After the initial immunization, booster immunizations were administered every 14 days at a dose of 0.1 mg, using the same emulsification method. A total of 5 immunizations were performed. Starting from the third booster immunization, 30 μL of blood was collected from the mouse tail 7 days after each immunization. The antiserum was collected by centrifugation and stored at -20°C for serum titer and specificity assays.

[0106] S2, ELISA indirect enzyme-linked immunosorbent assay for antiserum efficacy analysis

[0107] Using conventional antibody titer determination methods, with blank serum without antibodies as a control, the antiserum was diluted a certain number of times and then subjected to ELISA detection. The final result showed that the titer of the present invention's anti-N-acetyl-5-hydroxytryptamine specific antibody was 1:30000-1:50000, indicating that the antibody prepared by the present invention has high specificity and high sensitivity.

[0108] S3, cell fusion, and screening for positive hybridomas

[0109] A1. Resuscitating myeloma cells: Remove myeloma cells from liquid nitrogen and quickly place them in a 37°C water bath to thaw. After thawing, centrifuge at 1000 rpm for 5 minutes. Discard the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Disperse the cells by blowing them away. Use a pipette to remove the cells and mix them with the complete culture medium. Place the mixture into 10 cm diameter culture dishes and expand to 4-6 dishes. Change the medium several times during this process. When the cells in each culture dish cover the bottom, the cells can be used for cell fusion.

[0110] A2. Cell Preparation: Take two small culture dishes. Pour some culture medium into one dish to cool the dissecting tools, and aspirate a small amount of culture medium into the other dish and place it in a cell strainer for grinding the spleen. Transfer the revived myeloma cells to a 50mL centrifuge tube, seal the tube, and centrifuge at 1200 rpm for 5 minutes. Sacrifice the mice after five immunizations, soak them in 75% alcohol for about 1 minute, place them in a clean bench, collect blood from the heart, incubate at 37℃ for 30 minutes, centrifuge for 15 minutes, and store the serum at -20℃. After collecting the mouse spleen cells, grind them thoroughly in a cell strainer, wash them with pre-allocated basal solution, and transfer them to a 50mL centrifuge tube. Seal the tube. Discard the supernatant from the centrifuged myeloma cells, add basal solution and wash again, then centrifuge together with the spleen cells at 1200 rpm for 5 minutes. After the second centrifugation, discard the supernatant from the myeloma cells, add 2mL of basal solution, and mix well. Spleen cells were cleaned of supernatant, passed through a cell sieve, and added to myeloma cells. After mixing thoroughly by pipetting, basal culture medium was added to a final volume of 20 mL, and the mixture was centrifuged at 1200 rpm for 5 minutes. The centrifuged mixture was then removed, the supernatant was discarded, excess culture medium was aspirated, and the precipitated cells were dispersed by shaking. The mixture was then incubated at 37°C for 5 minutes.

[0111] A3. Cell Fusion: After incubation, place the centrifuge tube in 37°C warm water and keep it rotating throughout the process. Use a pipette tip to draw 1 mL of preheated PEG (37°C) and slowly add it to the precipitated cells within the first minute. Let it stand for 1 minute, preheat the basal medium, add 1 mL within the third minute, 3 mL within the fourth minute, and 16 mL within the fifth and sixth minutes, while gently stirring and adding it along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900 rpm for 8 minutes. Discard the supernatant and add the fused cells to HAT complete medium. Gently stir and evenly distribute the mixture into four 24-well culture plates. Ensure that the volume of HAT medium containing fused cells is the same in each well.

[0112] A4. Screening of positive hybridomas: Within 4 days after fusion, replace half of the medium with HT medium. After 8 days, replace all of the medium with HT medium in each well. On day 10, collect the supernatant from the multi-well culture plate and detect specific antibodies in the culture medium using indirect ELISA. Select positive hybridoma cells with high titers and strong affinity, and identify the positive wells with the best fusion effect, marking them accordingly. Under aseptic conditions, transfer to new 96-well culture plates, cloning each original well into two 96-well plates. After the cells adhere and grow to 1 / 4 of the bottom of the well, collect the supernatant and detect them by ELISA, again using titer and inhibition rate as indicators. Select cells with strong positive results and subclone them using limiting dilution. Repeat this process 3-4 times (note that the positive cells selected in each round need to be expanded in culture and then frozen for later use) until every well in every plate is positive and the titer and inhibition rate are similar. At this point, the hybridoma cell line has been successfully established, obtaining a hybridoma cell line that stably secretes uniform antibodies. Single-cell clones were selected, and those that tested positive for all cells were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10cm cell culture dishes for further culture and then frozen in time.

[0113] S4. Large-scale preparation of monoclonal antibodies

[0114] After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are typically prepared in large quantities using in vitro culture and in vivo animal induction methods. Liquid sclerosing agent (0.5 mL / mouse) was injected intraperitoneally into more than ten Balb / c mice over 8 weeks old beforehand. Hybridoma cells were injected intraperitoneally into the mice 1-2 weeks later. The mice were observed daily after cell inoculation, especially from day 7 onwards, when the peritoneum would swell. Ascites fluid was aseptically collected using a disposable syringe before the mice died. The collected ascites fluid was centrifuged at 12000 rpm for 10 minutes to remove the upper fat and lower fibrin layers, collecting the middle layer. Its titer and inhibition rate were determined using ELISA. After purification, it was stored at -20℃ for later use, yielding N-acetyl-5-hydroxytryptamine monoclonal antibody.

[0115] Example 9: N-acetyl-5-hydroxytryptamine ELISA test

[0116] The titer and specificity of the antiserum were determined using an indirect enzyme-linked immunosorbent assay (ELISA). The steps are as follows:

[0117] The antibody prepared in Example 8 was used for an ELISA test of N-acetyl-5-hydroxytryptamine. This test utilizes a competitive immunoassay to determine the N-acetyl-5-hydroxytryptamine content in a liquid sample. The principle is that N-acetyl-5-hydroxytryptamine in the sample competes with a conjugated N-acetyl-5-hydroxytryptamine derivative (HRP-N-acetyl-5-hydroxytryptamine derivative enzyme conjugate) for binding to a limited number of sites on the antibody coated in the ELISA plate. If the liquid sample contains little or no N-acetyl-5-hydroxytryptamine, the HRP-conjugated N-acetyl-5-hydroxytryptamine derivative will bind to the antibody in the ELISA plate. Conversely, if the liquid sample contains a large amount or a certain quantity of N-acetyl-5-hydroxytryptamine, the binding of the enzyme-conjugated N-acetyl-5-hydroxytryptamine derivative conjugate to the antibody will decrease, thus weakening the colorimetric signal. Therefore, the absorbance produced by the test is inversely proportional to the N-acetyl-5-hydroxytryptamine content in the liquid sample. The specific steps are as follows:

[0118] Establishment of a standard curve for N-acetyl-5-hydroxytryptamine ELISA detection

[0119] S1. Preparation of Standards

[0120] N-acetyl-5-hydroxytryptamine powder (purchased from Merck) was dissolved in methanol to prepare a stock solution of 1 mg / mL. The stock solution was then sequentially diluted with ELISA buffer to prepare standard solutions of 600 ng / mL, 200 ng / mL, 66.7 ng / mL, 22.2 ng / mL, 7.4 ng / mL, 2.4 ng / mL, 0.8 ng / mL, and 0 μng / mL. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl, and 0.2% BSA.

[0121] S2. Prepare a standard curve using the ELISA method for N-acetyl-5-hydroxytryptamine.

[0122] S3. Dilute the anti-N-acetyl-5-hydroxytryptamine antibody prepared in Example 7 to a final concentration of 1:10000 with PBS, and coat 100 μL / well of a 96-well ELISA plate. Incubate at 4°C for 12-24 h. Wash the 96-well ELISA plate coated with the anti-N-acetyl-5-hydroxytryptamine antibody three times with PBS, add 200 μL / well of 0.5% BSA solution, and block at 4°C for 8-16 h. Then wash three times with PBS and add 20 μL / well of standard. Add 100 μL / well of HRP-N-acetyl-5-hydroxytryptamine conjugate at the working concentration. Incubate at room temperature for 30 min, then wash the plate five times with PBS. Add 100 μL of TMB substrate to each well and incubate at room temperature for 30 min. Add 100 μL of stop solution (2M sulfuric acid) to each well. Measure the absorbance at 450 nm. Calibration was performed based on the absorbance values ​​at 450 nm corresponding to each standard sample, and a standard curve was constructed. The results are shown in the attached figure. Figure 1 As shown.

[0123] Example 10: Preparation of a chemiluminescent detection reagent for 5-hydroxytryptamine alkaline phosphatase

[0124] Preparation of a chemiluminescent detection reagent for 5-hydroxytryptamine alkaline phosphatase using the antibody obtained in Example 8.

[0125] S1. A 5-hydroxytryptamine (5-HT) detection kit, wherein sample preparation (converting 5-HT to N-acetyl-5-HT) is part of sample dilution, and each sample is incubated with an acetylation reagent to obtain acetylated 5-HT. This experiment uses the principle of competitive chemiluminescent immunoassay, where the analyte in the sample and the antigen labeled with a luminescent marker competitively bind to a certain number of antibody binding sites. The amount of antigen bound to the antibody is inversely proportional to the concentration of the analyte in the sample. After the reaction system reaches equilibrium, a substrate solution is added to detect the amount of antigen labeled with a luminescent marker bound to the antibody. A standard curve is plotted using known standards, and the concentration of 5-HT in the unknown sample is obtained by calibrating the luminescence value of the unknown sample on the standard curve. The kit is characterized by comprising a sample processing solution, chemiluminescently labeled N-acetyl-5-HT, and an N-acetyl-5-HT monoclonal antibody coated on a fixed carrier.

[0126] The sample processing solution includes an acetylation reagent and an organic solvent;

[0127] The sample processing solution contains an acetylation reagent at a concentration of 0.001–100 mg / mL, preferably 20–50 mg / mL; the acetylation reagent is one or more of acetyl chloride, acetic anhydride, and glacial acetic acid, preferably acetyl chloride; the organic solvent is one or more of benzene, acetone, ethanol, diethyl ether, and ethyl acetate, preferably ethanol. S2, the chemiluminescent label in the N-acetyl-5-hydroxytryptamine is alkaline phosphatase.

[0128] S3. The antibody coated on the immobilization carrier is an anti-N-acetyl-5-hydroxytryptamine antibody coated on magnetic beads.

[0129] Obtaining the standard curve: Set the reaction parameters for the alkaline phosphatase chemiluminescence analyzer (see Table 1). The instrumentation procedure is as follows: First, add the sample processing solution and sample (or standard), incubate at 37°C for 10 minutes; then add reagent 1 and reagent 2, react at 37°C for 5 minutes. Add the luminescent substrate solution, measure the luminescence value, and the machine automatically calculates the fitted calibration curve and calculates the sample concentration. The calibration curve is attached. Figure 2 As shown.

[0130] Table 1: Reaction parameters of alkaline phosphatase chemiluminescence analyzer

[0131]

[0132] The chemiluminescent detection reagent for 5-hydroxytryptamine alkaline phosphatase of this invention was compared with 5-hydroxytryptamine ELISA detection reagents from well-known foreign manufacturers. The detection data and data analysis are attached. Figure 3 .

[0133] Example 11: Cross-reactivity test of analogues

[0134] Nine common N-acetyl-5-hydroxytryptamine structural analogs were selected for cross-reactivity testing, and the results were determined using an alkaline phosphatase chemiluminescence assay. The nine common N-acetyl-5-hydroxytryptamine structural analogs and their cross-reactivity rates are shown in Table 2.

[0135] Table 2: Cross-reactivity results of 9 common N-acetyl-5-hydroxytryptamine structural analogues

[0136] 5-hydroxytryptamine Cross-reactivity (%) N-acetylserotonin 100 5-Methoxytryptamine 0.1 5-HydroxyL-Tryptophan 0.04 melatonin 0.01 Pentahydroxyindoleacetic acid 0 L-Tryptophan 0.01 Indole-3-acrylic acid 0 Indole-3-pyruvic acid 0.01

[0137] Results: Based on the cross-reactivity results of the above nine common N-acetyl-5-hydroxytryptamine structural analogs, the antibody prepared in this invention has good specificity.

[0138] The results above show that the N-acetyl-5-hydroxytryptamine complete immunogen prepared from the N-acetyl-5-hydroxytryptamine derivative provided by this invention has strong immunogenicity, produces antibodies with high specificity and good affinity for N-acetyl-5-hydroxytryptamine, and the alkaline phosphatase chemiluminescence reagent prepared using the above antibody has good stability and high sensitivity. It can realize high-throughput and rapid detection of serotonin on a fully automated chemiluminescence analyzer, and has the advantages of simple operation, high sensitivity, strong specificity, and accurate results. It can also effectively reduce the detection cost of N-acetyl-5-hydroxytryptamine, which is conducive to its clinical application.

[0139] It should be noted that the above description is only an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An N-acetyl-5-hydroxytryptamine derivative, characterized in that, It has the structural formula shown in equation (Ⅰ): Where R is a linking group, R is —(CH2) n —COOH, where n is an integer between 1, 2, and 3.

2. An N-acetyl-5-hydroxytryptamine immunogen, characterized in that, It has the structural formula shown in equation (Ⅱ): Where R is —(CH2) n —COOH, n is an integer between 1, 2, and 3, and the carrier is an immunogenic protein or polypeptide; the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine.

3. A specific antibody against N-acetyl-5-hydroxytryptamine, produced by immunizing animals with an immunogen, characterized in that, The anti-N-acetyl-5-hydroxytryptamine specific antibody was obtained by immunizing animals with the N-acetyl-5-hydroxytryptamine immunogen described in claim 2.

4. The antibody according to claim 3, characterized in that, The antibody is a complete protein molecule or a polypeptide fragment capable of specifically binding to the N-acetyl-5-hydroxytryptamine immunogen.

5. The antibody according to claim 3, characterized in that, The antibody is a polyclonal antibody, a monoclonal antibody, or a recombinant antibody.

6. The antibody according to claim 3, characterized in that, The antibody was obtained by immunizing rabbits, goats, mice, sheep, guinea pigs, horses, alpacas, or camels with the N-acetyl-5-hydroxytryptamine immunogen.

7. A method for preparing an N-acetyl-5-hydroxytryptamine immunogen, characterized in that, The preparation method includes: preparing the N-acetyl-5-hydroxytryptamine derivative according to claim 1; and linking the N-acetyl-5-hydroxytryptamine derivative to a carrier to obtain the N-acetyl-5-hydroxytryptamine immunogen; wherein the carrier is an immunogenic protein or polypeptide, and the step of preparing the N-acetyl-5-hydroxytryptamine derivative includes: S1. N-acetyl-5-hydroxytryptamine was subjected to a substitution reaction with halo-(CH2)n-COO-tert-butyl ester in an N,N-dimethylformamide (DMF) environment to obtain 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine; S2. Dissolve the 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain a reaction solution. Stir the reaction solution at 40-60°C for 2.5-6.5 h to obtain the N-acetyl-5-hydroxytryptamine derivative.

8. The preparation method according to claim 7, characterized in that, Step S1 includes the following steps: A1. N-acetyl-5-hydroxytryptamine and halo-(CH2)n-COO-tert-butyl ester are dissolved in diN,N-dimethylformamide (DMF) to obtain a mixture; wherein the molar ratio of N-acetyl-5-hydroxytryptamine to halo-(CH2)n-COO-tert-butyl ester is 0.8 to 1.6:1; the temperature at which the mixture is heated to a constant temperature is 70 to 100°C, and the time for which the mixture is kept at a constant temperature is greater than or equal to 32 hours; A2. Add ethyl acrylate to the mixture to obtain a reaction mixture, and stir the reaction mixture at a low temperature of 0-8°C to obtain a solid-liquid mixture; A3. The reaction mixture is filtered, washed, purified, and vacuum dried to obtain the 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

9. The preparation method according to claim 7 or 8, characterized in that, When n=1, the preparation steps of the N-acetyl-5-hydroxytryptamine derivative are as follows:

10. The preparation method according to claim 7, characterized in that, The coupling step between the carrier and the N-acetyl-5-hydroxytryptamine derivative includes: B1. Prepare a carrier solution and an N-acetyl-5-hydroxytryptamine derivative solution; wherein the carrier is serum protein, keyhole hemocyanin, thyroglobulin, or polylysine; B2. Add the N-acetyl-5-hydroxytryptamine derivative solution to the carrier solution to obtain a mixed solution of activated N-acetyl-5-hydroxytryptamine derivative and carrier; B3. Stir the mixed solution overnight at 2-10°C or react at room temperature for 2 hours to obtain the crude N-acetyl-5-hydroxytryptamine immunogen. B4. The crude N-acetyl-5-hydroxytryptamine immunogen is purified to obtain the pure N-acetyl-5-hydroxytryptamine immunogen.

11. The preparation method according to claim 7, characterized in that, The step of preparing the carrier solution includes dissolving the carrier in a 0.05–0.2 M phosphate buffer solution with a pH of 8.0–9.5 to obtain the carrier solution; and the step of preparing the N-acetyl-5-hydroxytryptamine derivative solution includes placing the N-acetyl-5-hydroxytryptamine derivative, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDAc), and N-hydroxythiosuccinimide (Sulfo-NHS) in N,N-dimethylformamide, methanol, and a 5–20 mM phosphate buffer solution with a pH of 4.0–6.0 at room temperature and stirring to obtain a solution of the N-acetyl-5-hydroxytryptamine derivative in an activated state; wherein the mass ratio of the carrier to the N-acetyl-5-hydroxytryptamine derivative is 1–8:

1.

12. A 5-hydroxytryptamine detection kit, characterized in that, The kit contains the N-acetyl-5-hydroxytryptamine derivative of claim 1 and the antibody of any one of claims 3 to 6.

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