Preparation and application of N-acetyl-5-hydroxytryptamine derivative, immunogen, antibody and detection reagent

Abstract

5-hydroxytryptamine (5-HT) is found from serum at the earliest and is also known as serotonin, and due to the fact that the 5-hydroxytryptamine is small in molecular weight and poor in immunogenicity, the specificity and affinity of a prepared antibody cannot meet the requirements of an immunodetection reagent. According to the invention, N-acetyl-5-hydroxytryptamine with large molecular weight is innovatively used for hapten design, and through coupling with a specific macromolecular carrier, the prepared complete antigen has the characteristics of good specificity, strong affinity and the like. The invention mainly relates to design and synthesis of an N-acetyl-5-hydroxytryptamine hapten, an N-acetyl-5-hydroxytryptamine complete antigen, preparation of an anti-N-acetyl-5-hydroxytryptamine antibody, a method for determining the concentration of 5-hydroxytryptamine, and composition and components of a detection reagent, and belongs to the technical field of biomedical detection. The N-acetyl serotonin derivative disclosed by the invention has a structure as shown in a formula (I): # imgabs0 #, wherein R is-(CH2) n-COOH, and n is an integer between 1 and 10.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, and in particular to an N-acetyl-5-hydroxytryptamine derivative, an N-acetyl-5-hydroxytryptamine immunogen and a specific antibody thereof, and a 5-hydroxytryptamine detection kit. Background Art

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

[0003]

[0004] 5-HT is an intermediate product of tryptophan metabolism. It 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 5-HT in the circulating blood is concentrated in platelets. Changes in the concentration of circulating 5-HT can reflect some pathological changes in the human body, including chronic tension headaches, schizophrenia, hypertension, chorea, Duchenne muscular dystrophy and early acute appendicitis. In addition, the detection of serum 5-HT is also of great clinical significance for the detection of carcinoid syndrome.

[0005] At present, the main methods for 5-HT detection are fluorescence analysis, enzyme-linked immunosorbent assay, gas chromatography, and high-performance liquid chromatography. The above methods are cumbersome to operate, time-consuming and labor-intensive, and cannot take into account both accuracy and timeliness in clinical tests. The present invention starts with antigen design and antibody preparation, and adopts an advanced fully automatic chemiluminescence detection platform to completely solve the pain points of 5-HT clinical testing, and has broad application prospects. This detection reagent can be used for high-throughput, automated, and multi-type sample determination, and has the outstanding advantages of high sensitivity, specificity, and good stability in reagent performance. Summary of the invention

[0006] The purpose of the present 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 to improve the defects of the prior art 5-hydroxytryptamine determination method, such as poor specificity, low sensitivity and inability to perform automated analysis.

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

[0008]

[0009] Wherein, R is a connecting group -(CH2)n-COOH, and n is an integer between 1 and 10. The N-acetyl-5-hydroxytryptamine derivative of the present invention has a basic structure for preparing an N-acetyl-5-hydroxytryptamine immunogen with immunogenicity, and provides a structural basis for preparing a 5-hydroxytryptamine detection reagent.

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

[0011] Formula (II)

[0012] Wherein R is a connecting 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 the present invention has high immunogenicity, can stimulate an animal body to produce an immune response, produce a high-titer anti-N-acetyl-5-hydroxytryptamine specific antibody, and has a strong antibody affinity, and is suitable for preparing a highly sensitive and highly specific 5-hydroxytryptamine competitive detection reagent.

[0013] When n=1, R in the above N-acetyl-5-hydroxytryptamine immunogen is -CH2-COOH. The carrier suitable for the above N-acetyl-5-hydroxytryptamine immunogen is preferably a protein carrier, but other types of immunogenic substances with a sufficiently large molecular weight and sufficient active groups can also be used as carriers. The most commonly used immunogenic carriers include serum protein, keyhole limpet hemocyanin (KLH), thyroglobulin and polylysine. The carrier in the present invention is preferably keyhole limpet hemocyanin.

[0014] According to another aspect of the present invention, there is also provided an anti-N-acetyl-5-hydroxytryptamine specific antibody, which is produced by immunizing an animal with an immunogen, and the antibody is produced by immunizing an animal with any of the above-mentioned N-acetyl-5-hydroxytryptamine immunogens. The "antibody" referred to in the present invention refers not only to a complete protein molecule antibody, but also includes a polypeptide fragment antibody or a derivative of a polypeptide fragment antibody that retains the specific binding ability of the complete antibody. The antibody of the present invention can be a polyclonal antibody, a monoclonal antibody or a recombinant antibody, preferably a monoclonal antibody.

[0015] The antibodies of the present invention can be prepared by existing techniques. A typical method for obtaining polyclonal antibodies is to use a single immunogen, with or without an adjuvant, to immunize one or more parts of an animal, wherein the host animal includes: rabbits, goats, mice, sheep, guinea pigs, horses, alpacas or camels. Immunization is continued for 5-7 times until the antibody titer reaches the highest. Blood is collected from the animal at regular intervals to obtain an appropriate amount of specific antiserum. Monoclonal antibodies can be prepared by hybridoma cell technology. Recombinant antibodies can be prepared by genetic engineering expression technology.

[0016] According to another aspect of the present invention, there is also provided a method for preparing an N-acetyl-5-hydroxytryptamine immunogen, the method comprising the steps of preparing the above-mentioned N-acetyl-5-hydroxytryptamine derivative and connecting the above-mentioned N-acetyl-5-hydroxytryptamine derivative with a carrier to obtain an N-acetyl-5-hydroxytryptamine immunogen. The N-acetyl-5-hydroxytryptamine derivative prepared by the above-mentioned method is connected with an immunogenic protein or polypeptide to obtain the N-acetyl-5-hydroxytryptamine immunogen with strong immunogenicity of the present invention, and the preparation method is simple to operate.

[0017] In the method for preparing the above-mentioned N-acetyl-5-hydroxytryptamine immunogen of the present invention, a method for preparing the above-mentioned N-acetyl-5-hydroxytryptamine derivative is also provided, and the preparation method comprises the following steps: S1, subjecting N-acetyl-5-hydroxytryptamine 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; S2. Dissolve 2-(CH2)n-COO-tert-butyl N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain an N-acetyl-5-hydroxytryptamine derivative. The preparation method replaces the hydrogen at the 5-position of N-acetyl-5-hydroxytryptamine with a halogenated -(CH2)n-COO-tert-butyl ester to generate 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine, and then in an acidic environment, opens the 2-(CH2)n-COO-tert-butyl ester bond on the 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine to form an N-acetyl-5-hydroxytryptamine derivative having -(CH2)n-COOH at the 5-position of N-acetyl-5-hydroxytryptamine. The preparation method is simple to operate, has mild reaction conditions, high stability, and good repeatability.

[0018] In the above-mentioned preparation step S1 of the N-acetyl-5-hydroxytryptamine derivative, the following steps are also included: A1, dissolving N-acetyl-5-hydroxytryptamine and halo-(CH2)n-COO-tert-butyl ester in N,N-dimethylformamide (DMF) to obtain a mixed solution; A2, adding ethyl acrylate to the mixed solution to obtain a solid-liquid mixture; A3. Filter the solid-liquid mixture to obtain 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine. N-acetyl-5-hydroxytryptamine and halogenated-(CH2)n-COO-tert-butyl ester have good solubility in DMF organic solvent. Ethyl acrylate is added to precipitate 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine, and the reactant is filtered to obtain.

[0019] In the above step A1, after the mixed solution is obtained, the mixed solution is further heated to a constant temperature, preferably at a temperature of 70 to 100° C. for a constant temperature time of greater than or equal to 32 hours. Heating the mixed solution to a constant temperature for more than 32 hours can ensure sufficient dissolution and reaction.

[0020] In the present invention, in the above step A2, the reaction mixture is stirred and solidified at a low temperature of 0 to 8° C. to increase the yield.

[0021] In the preparation method of the above-mentioned N-acetyl-5-hydroxytryptamine derivative of the present invention, the specific operation of filtering the solid-liquid mixture to obtain 2-(CH2)-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine has no special requirements, as long as the desired target product can be separated from the solid-liquid mixture. In the present invention, the above-mentioned 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 ester N-acetyl-5-hydroxytryptamine. After the steps of filtering, washing, purifying and vacuum drying, the purity and yield of the obtained 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine are relatively high.

[0022] In the preparation method of the above-mentioned N-acetyl-5-hydroxytryptamine derivatives of the present invention, the specific operation process of the step of dissolving 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain the N-acetyl-5-hydroxytryptamine derivative can be appropriately adjusted according to the difference between 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine and the acid solution. In the present invention, preferably, the above-mentioned step S2 includes: dissolving 2-(CH2)n-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine in a strong acid solution to obtain a reaction solution; stirring the reaction solution at 40 to 60° C. for 2.5 to 6.5 hours to obtain a stirred solution; drying the stirred solution to obtain a dried product; washing and purifying the dried product with acetone to obtain an N-acetyl-5-hydroxytryptamine derivative. The production of the target N-acetyl-5-hydroxytryptamine derivative can be promoted by stirring at a high temperature of about 60°C, and a dried product containing the target N-acetyl-5-hydroxytryptamine derivative is obtained by evaporation and drying. After washing with acetone, the organic residue on the dried product is removed to obtain the target N-acetyl-5-hydroxytryptamine derivative with higher purity.

[0023] 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:

[0024]

[0025] When n=1, the reaction steps are the same as above, except that tert-butyl bromoacetate is used as the synthetic raw material, so the linking group R of the final product N-acetyl-5-hydroxytryptamine derivative is -CH2-COO-.

[0026] In the method for preparing the above-mentioned N-acetyl-5-hydroxytryptamine immunogen of the present invention, the step of connecting the carrier and the N-acetyl-5-hydroxytryptamine derivative can be reasonably improved according to the different carriers in actual operation. In the present invention, the above-mentioned connecting step includes: S1, preparing 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 limpet hemocyanin, thyroglobulin or poly-lysine; S2, adding the activated N-acetyl-5-hydroxytryptamine derivative solution dropwise to the carrier solution to obtain a crude N-acetyl-5-hydroxytryptamine immunogen; S3, stirring the added mixed solution at 2-10° C. overnight or reacting at room temperature for 2 hours to obtain a crude N-acetyl-5-hydroxytryptamine immunogen; S4, purifying the crude N-acetyl-5-hydroxytryptamine immunogen to obtain the N-acetyl-5-hydroxytryptamine immunogen. The preparation steps of the present 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.

[0027] In the method for preparing the above-mentioned N-acetyl-5-hydroxytryptamine immunogen of the present invention, in the actual operation of the steps of preparing the carrier solution and preparing the N-acetyl-5-hydroxytryptamine derivative solution, the appropriate solvent concentration and pH value are reasonably selected according to the different types of carriers. In the above-mentioned step S1 of the present invention, the step of preparing the carrier solution is to dissolve the carrier in a phosphate buffer of 0.05-0.20M and pH 8.0-9.5 to obtain a carrier solution; the step of preparing the N-acetyl-5-hydroxytryptamine derivative solution is to place the N-acetyl-5-hydroxytryptamine derivative, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide in N,N-dimethylformamide, methanol and 5-20mM, pH 4.0-6.0 potassium phosphate buffer and stir at room temperature to obtain an N-acetyl-5-hydroxytryptamine derivative activated state solution. The carrier is dissolved in a phosphate buffer solution with a concentration of 0.10 to 0.25 M and a pH of 8.0 to 9.5, so that the amino group on the carrier and the activated carboxyl group of the N-acetyl-5-hydroxytryptamine derivative can fully react and combine in a slightly alkaline environment. The carboxyl group of the N-acetyl-5-hydroxytryptamine derivative can be fully activated in a slightly acidic solution by using a phosphate buffer solution with a concentration of 5 to 20 mM and a pH of 4.0 to 6.0.

[0028] In the method for preparing the N-acetyl-5-hydroxytryptamine immunogen of the present invention, in the step of dripping to obtain the crude N-acetyl-5-hydroxytryptamine immunogen, in order to further increase the content of the N-acetyl-5-hydroxytryptamine immunogen in the crude N-acetyl-5-hydroxytryptamine immunogen, in the steps S2 and S3 of the present invention, the dripping step can make the N-acetyl-5-hydroxytryptamine derivative react more fully with the carrier; stirring overnight at 2 to 10° C. can further promote the generation of the N-acetyl-5-hydroxytryptamine immunogen, thereby increasing the content of the N-acetyl-5-hydroxytryptamine immunogen in the crude product.

[0029] In the above-mentioned method for preparing N-acetyl-5-hydroxytryptamine immunogen of the present invention, any operation that can purify the crude N-acetyl-5-hydroxytryptamine immunogen to obtain the N-acetyl-5-hydroxytryptamine immunogen is applicable to the present invention. Preferably, in the above-mentioned step S4, the crude N-acetyl-5-hydroxytryptamine immunogen is purified by dialysis to obtain the N-acetyl-5-hydroxytryptamine immunogen, and the dialysis method is simple and has a good purification effect.

[0030] According to another aspect of the present invention, there is also provided an N-acetyl-5-hydroxytryptamine detection reagent, 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 above-mentioned anti-N-acetyl-5-hydroxytryptamine specific antibodies; the N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate contains the above-mentioned N-acetyl-5-hydroxytryptamine derivative, and the N-acetyl-5-hydroxytryptamine enzyme-labeled conjugate is formed by coupling an enzyme and a hapten, wherein the hapten is the above-mentioned N-acetyl-5-hydroxytryptamine derivative.

[0031] The above-mentioned N-acetyl-5-hydroxytryptamine detection reagent of the present invention has a high specificity of the anti-N-acetyl-5-hydroxytryptamine specific antibody and a strong binding force with N-acetyl-5-hydroxytryptamine, and the detection sensitivity is much higher than the corresponding products in the prior art. Preferably, the above-mentioned enzyme-labeled conjugate is an alkaline phosphatase-hapten enzyme-labeled conjugate; and the substrate of the above-mentioned enzyme is AMPPD or APS-5. The detection reagent using the 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 the sample, and is suitable for high-throughput automated detection.

[0032] According to another aspect of the present invention, a 5-hydroxytryptamine detection kit is also provided, which contains the above-mentioned anti-N-acetyl-5-hydroxytryptamine specific antibody, 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 an enzyme reagent, a radioisotope reagent, a fluorescent reagent, and a luminescent reagent. Preferably, the indicator reagent is composed 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 to the N-acetyl-5-hydroxytryptamine derivative of the present invention, and the N-acetyl-5-hydroxytryptamine content in the sample can be conveniently and accurately determined, and is suitable for high-throughput automated detection.

[0033] By using the technical solution of the present invention, the N-acetyl-5-hydroxytryptamine derivative obtained by replacing the hydrogen at a specific site with a specific R1 group is connected to a specific carrier to form an N-acetyl-5-hydroxytryptamine immunogen, which has high immunogenicity, high specificity of antibodies produced by immune-induced animals, and strong specific binding ability with N-acetyl-5-hydroxytryptamine. High-throughput and rapid detection of N-acetyl-5-hydroxytryptamine on a fully automatic chemiluminescence immunoassay can be achieved by using alkaline phosphatase chemiluminescence immunoassay technology, and has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc., and can also effectively reduce the cost of N-acetyl-5-hydroxytryptamine detection, which is conducive to clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Attached Figure 1: Standard curve of N-acetyl-5-hydroxytryptamine ELISA detection.

[0035] Attached Figure 2 : Calibration curve of chemiluminescence of N-acetyl-5-hydroxytryptamine alkaline phosphatase.

[0036] Attached Figure 3 : Sample comparison results of the N-acetyl-5-hydroxytryptamine chemiluminescent detection reagent of the present invention and the ELISA detection reagent of the comparison manufacturer. DETAILED DESCRIPTION

[0037] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0038] Example 1: Synthesis and structural confirmation of N-acetyl-5-hydroxytryptamine derivatives

[0039] The chemical structure of the N-acetyl-5-hydroxytryptamine derivatives used in the following examples is shown in formula (IV):

[0040]

[0041] The specific synthesis steps of the N-acetyl-5-hydroxytryptamine derivative represented by formula (IV) are as follows:

[0042] S1. Synthesis of Compound 2

[0043] The specific synthesis steps of compound 2 are as follows:

[0044] 15.0 g of compound 1 and 26 g of tert-butyl bromoacetate were weighed and dissolved in 150 mL of N,N-dimethylformamide (DMF), and then the solution was heated to 95°C overnight. 500 mL of ethyl acrylate (EA) was added to the solution, and the reaction mixture was stirred at 0°C for 25 min. The solid precipitate in the solution was filtered out, washed with acetone, and then vacuum dried to finally obtain 11.2 g of brown solid compound 2 with a yield of 30%.

[0045] S2. Synthesis of N-acetyl-5-hydroxytryptamine derivatives

[0046]

[0047] A1. Weigh 3.00 g of compound 2, dissolve it in 30 mL of HCl (1.5 M), and stir the solution at 60° C. for 3 hours. Evaporate the reaction mixture to dryness, and wash the dried residue with acetone to obtain 2.10 g of a reddish brown solid N-acetyl-5-hydroxytryptamine derivative with a yield of 92%.

[0049] A2. The compound was subjected to nuclear magnetic resonance spectroscopy scanning using Varian III plus 300 MHz, with TMS used as an internal standard. The result was characterized as 2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0048] Example 2: Synthesis and preparation of derivatives when n=2

[0049] S1. Synthesis of 2-(CH2)2-COO-tert-butyl N-acetyl-5-hydroxytryptamine

[0050] A1. Weigh 18.0g of compound 1 and 27.8g of tert-butyl bromopropionate, dissolve them in 150mL of N,N-dimethylformamide (DMF), and then heat the solution to 95°C overnight. Add 550mL of ethyl acrylate (EA) to the solution, and stir the reaction mixture at 4°C for 25min. Filter the solid precipitate in the solution, wash it with acetone, and then vacuum dry it to finally obtain 7.70g of brown solid intermediate product with a yield of 26%.

[0051] A2. The compound was subjected to nuclear magnetic resonance spectroscopy scanning using Varian III plus 300 MHz, with TMS used as an internal standard. The result was characterized as 2-(CH2)2-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0052] S2. Synthesis of N-acetyl-5-hydroxytryptamine derivatives

[0053] A1. Weigh 3.00 g of the above 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 dried residue with acetone to obtain 2.1 g of a reddish brown solid N-acetyl-5-hydroxytryptamine derivative with a yield of 92.1%.

[0054] A2. Structural identification of the purified product obtained above

[0055] a. The compound was subjected to nuclear magnetic resonance spectroscopy scanning using Varian III plus 300 MHz, with TMS used as the internal standard. The result was characterized as 2-(CH2)2-COO-N-acetyl-5-hydroxytryptamine.

[0056] b. The obtained derivatives were analyzed and identified using chromatography / mass spectrometry (LC / MS) technology, and it was determined that the final compound was 2-(CH2)2-COO-N-acetyl-5-hydroxytryptamine.

[0057] Example 3: Synthesis and preparation of N-acetyl-5-hydroxytryptamine derivatives when n=3

[0058] S1. Synthesis of 2-(CH2)3-COO-tert-butyl N-acetyl-5-hydroxytryptamine

[0059] A1. Weigh 18.0 g of compound 1 and 29.5 g of tert-butyl bromobutyrate, dissolve them in 180 mL of N,N-dimethylformamide (DMF), and then heat the solution to 95°C overnight. Add 525 mL of ethyl acrylate (EA) to the solution, and stir the reaction mixture at 5°C for 30 min. Filter the solid precipitate in the solution, wash it with acetone, and then vacuum dry it to finally obtain 7.2 g of compound 2 as a white solid with a yield of 22%.

[0060] A2. The compound was subjected to nuclear magnetic resonance spectroscopy scanning using Varian III plus 300 MHz and TMS was used as an internal standard. The result showed that the compound was characterized as 2-(CH2)3-COO-tert-butyl ester N-acetyl-5-hydroxytryptamine.

[0061] S2. Synthesis of N-acetyl-5-hydroxytryptamine derivatives

[0062] A1. Weigh 3.00 g of compound 2, dissolve it in 30 mL of HCl (1.5 M), and stir the solution at 55° C. for 4.5 hours. Evaporate the reaction mixture to dryness, and wash the dried residue with acetone to obtain 2.1 g of a brown solid N-acetyl-5-hydroxytryptamine derivative with a yield of 94%.

[0063] A2. Structural identification of the purified product obtained above

[0064] a. The above compound was scanned by nuclear magnetic resonance spectrum using Varian III plus 300 MHz, with TMS as the internal standard. The result was characterized as 2-(CH2)3-COO-N-acetyl-5-hydroxytryptamine.

[0065] b. The obtained derivatives were analyzed and identified using chromatography / mass spectrometry (LC / MS) technology, and it was determined that the final compound was 2-(CH2)3-COO-N-acetyl-5-hydroxytryptamine.

[0066] In the above embodiments, when n=1, 2 and 3, tert-butyl bromoacetate, tert-butyl bromopropionate and tert-butyl bromobutyrate are respectively selected as synthetic raw materials in the synthesis of intermediate compounds in the preparation of N-acetyl-5-hydroxytryptamine derivatives, so the linking groups R1 of the obtained final products of N-acetyl-5-hydroxytryptamine derivatives are -CH2-COO-, -(CH2)2-COO- and -(CH2)3-COO-, respectively. When n is other integers from 1 to 10, other tert-butyl brominated organic acid esters similar to tert-butyl bromoacetate are selected for experiments, and the synthesis method is completely consistent except for the different values ​​of n.

[0067] Example 4: Synthesis of BSA-N-acetyl-5-hydroxytryptamine derivative immunogen

[0068] The BSA-N-acetyl-5-hydroxytryptamine immunogen is formed by connecting 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 the immunogen is described in detail by taking n=1 as an example. The specific steps are as follows:

[0069] S1. Dissolve bovine serum albumin (20 mg) in 5 ml of 0.2 M phosphate buffer, pH 8.5;

[0070] S2. Add the following chemicals into a small beaker and stir to dissolve: 20 mg of a synthetic 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-hydroxysulfosuccinimide (Sulfo-NHS), and stir to dissolve these chemicals at room temperature for 10 min.

[0071] S3. Add the activated solution dropwise into the BSA solution and stir overnight at 2-10°C to obtain a crude antigen product; purify the synthesized antigen by dialysis to obtain a 2-acetyl-5-hydroxytryptamine immunogen.

[0072] Example 5: Synthesis of KLH-N-acetyl-5-hydroxytryptamine derivative immunogen

[0073] The KLH-acetyl-5-hydroxytryptamine immunogen is formed by connecting 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 the immunogen is described in detail by taking n=2 as an example. The specific steps are as follows:

[0074] S1. Dissolve hemocyanin (20 mg) in 5 ml of 0.18 M phosphate buffer, pH 8.5;

[0075] S2. Add the following chemicals into a small beaker and stir to dissolve: 20 mg of a synthetic 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-hydroxysulfosuccinimide (Sulfo-NHS), and stir to dissolve these chemicals at room temperature for 10 min.

[0076] S3. Add the activated solution dropwise to the KLH solution and stir overnight at 2-10°C to obtain a crude N-acetyl-5-hydroxytryptamine immunogen; purify the synthesized antigen by dialysis to obtain a 2-propionic acid N-acetyl-5-hydroxytryptamine immunogen.

[0077] Example 6: Synthesis of thyroglobulin N-acetyl-5-hydroxytryptamine derivative immunogen

[0078] The thyroglobulin N-acetyl-5-hydroxytryptamine immunogen is formed by connecting 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 the immunogen is described in detail by taking n=3 as an example. The specific steps are as follows:

[0079] S1. Dissolve thyroglobulin (20 mg) in 5 ml of 0.20 M, pH 9.0 phosphate buffer;

[0080] S2. Add the following chemicals into a small beaker and stir to dissolve: 10 mg of a synthetic 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-hydroxysulfosuccinimide (Sulfo-NHS), and stir to dissolve these chemicals at room temperature for 30 min.

[0081] S3. Add the activated solution dropwise into the thyroglobulin solution and stir overnight at 2-10°C to obtain a complete antigen; purify the synthesized antigen by dialysis to obtain 2-butyric acid N-acetyl-5-hydroxytryptamine immunogen.

[0082] Example 7: Synthesis of poly-lysine-N-acetyl-5-hydroxytryptamine derivative immunogen

[0083] The poly-lysine-N-acetyl-5-hydroxytryptamine immunogen is formed by connecting poly-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 by taking n=3 as an example. The specific steps are as follows:

[0084] S1. Dissolve poly-lysine (20 mg) in 5 ml of 0.20 M, pH 9.0 phosphate buffer;

[0085] S2. Add the following chemicals into a small beaker and stir to dissolve: 10 mg of a synthetic 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-hydroxysulfosuccinimide (Sulfo-NHS), and stir to dissolve these chemicals at room temperature for 30 min.

[0086] S3. Add the activated solution dropwise into the poly-lysine solution and stir overnight at 2-10°C to obtain a complete antigen; centrifuge the synthesized antigen to obtain the supernatant, purify the supernatant by dialyzing to obtain 2-butyric acid N-acetyl-5-hydroxytryptamine immunogen.

[0087] Similarly, when n is other integers in the range of 1 to 10, the same method can be used to prepare N-acetyl-5-hydroxytryptamine immunogens as shown in formula (II), and there is no significant difference in the experimental results. The N-acetyl-5-hydroxytryptamine immunogens prepared using N-acetyl-5-hydroxytryptamine derivatives with different n values ​​all have strong immunogenicity, and the corresponding specific antibodies all have excellent performance. Of course, the carrier is still an immunogenic protein, which can be serum protein, keyhole limpet hemocyanin (KLH), thyroglobulin and polylysine. Preferably, the carrier is keyhole limpet hemocyanin.

[0088] Example 8: Preparation of anti-N-acetyl-5-hydroxytryptamine specific monoclonal antibody

[0089] This embodiment provides a method for preparing a monoclonal antibody of N-acetyl-5-hydroxytryptamine, which comprises the following steps:

[0090] S1. Animal Immunization The artificial antigen prepared in Example 5 was used to immunize two 8-week-old Balb / C female mice, and the immunization was performed by subcutaneous multiple-point injection at the back of the neck. The immunogen emulsified with Freund's complete adjuvant was used for the first immunization. The same volume of immunogen and Freund's complete adjuvant were mixed for emulsification, and the immunogen dose for a single mouse was 0.1 mg; after the first immunization, booster immunization was performed every 14 days, with a dose of 0.1 mg, and the emulsification method was the same; a total of 5 immunizations were performed, starting from the third booster immunization, and 7 days after each immunization, 30uL of blood was collected from the tail of the mouse, and the antiserum was centrifuged and stored at -20°C for serum titer determination and specificity determination.

[0091] S2. ELISA indirect enzyme-linked immunosorbent assay to analyze the effect of antiserum By using a conventional antibody titer determination method, blank serum without antibodies was used as a control, and the antiserum was diluted a certain multiple and then subjected to ELISA detection. The final detection showed that the titer of the anti-N-acetyl-5-hydroxytryptamine specific antibody of the present invention was 1:30000-1:50000, indicating that the antibody prepared by the present invention has strong specificity and high sensitivity.

[0092] S3. Cell fusion and screening of positive hybridomas

[0093] A1. Resuscitation of myeloma cells: Take out the myeloma cells from liquid nitrogen, quickly thaw them in a 37°C water bath, centrifuge at 1000r / min for 5 minutes after thawing, pour out the supernatant in an ultra-clean workbench, add about 1mL of complete culture medium to the cell pellet, blow away the cells, take out with a pipette and mix with complete culture medium, then put into a 10cm diameter culture dish and expand to 4 to 6 dishes. During this period, the liquid needs to be changed several times. When the cells in each culture dish cover the bottom, it can be used for cell fusion.

[0094] A2. Cell preparation: Take out two small culture dishes, pour part of the culture medium into one for cooling the dissection tools, and absorb a small amount of culture medium into the other and put it into a cell screen for grinding the spleen. Blow down the revived myeloma cells and transfer them to a 50mL centrifuge tube, seal it, and centrifuge it at 1200r / min for 5 minutes. The mice after five immunizations were killed, soaked in 75% alcohol for about 1 minute, placed in a clean bench, blood was taken from the heart, warmed at 37°C for 30 minutes, centrifuged for 15 minutes, and serum was taken and stored at -20°C. After taking the mouse spleen cells, grind them thoroughly in the cell screen, wash them with the pre-packaged basic solution, transfer them to a 50mL centrifuge tube, seal it, discard the supernatant of the centrifuged myeloma cells, add the basic solution to wash again, and centrifuge them at 1200r / min for 5 minutes with the spleen cells. After the second centrifugation, discard the supernatant of the myeloma cells, add 2mL of basic solution, and blow evenly. Remove the supernatant of spleen cells, add them to myeloma cells through a cell sieve, blow evenly, add basal culture medium to 20mL, and centrifuge at 1200r / min for 5 minutes. Take out the mixed cells after centrifugation and discard the supernatant, suck off the excess culture medium with a gun, shake the precipitated cells, and then incubate them in a 37℃ cell culture incubator for 5 minutes.

[0095] A3. Cell fusion: After the incubation, place the centrifuge tube in 37°C warm water, keep rotating throughout the process, use the gun tip to absorb 1mL of PEG preheated to 37°C, slowly add PEG to the precipitated cells within the first minute, let it stand for 1 minute, preheat the basal culture medium, add 1mL within the third minute, add 3mL within the fourth minute, and add 16mL within the fifth to sixth minutes, with gentle stirring, add along the wall to separate the PEG. Seal the centrifuge tube and centrifuge at 900r / min for 8 minutes, pour off the supernatant, add the fused cells to the HAT complete culture medium, stir gently, and evenly add to 4 24-well culture plates. Keep the volume of HAT culture medium containing fused cells in each well the same.

[0096] A4. Screening of positive hybridomas: Half of the medium was replaced with HT medium within 4 days after fusion, and the medium was fully replaced with HT medium in each well after 8 days. On the 10th day, the supernatant in the multi-well culture plate was extracted, and the specific antibodies in the culture medium were detected by indirect ELISA. The positive hybridoma cells with high titer and strong affinity were selected, and the positive wells with the best fusion effect were screened and marked. Under sterile conditions, the cells were transferred to a new 96-well culture plate, and each original well was cloned into two 96-well plates. After the cells adhered to the wall and covered 1 / 4 of the bottom of the well, the supernatant was taken and tested by ELISA. The titer and inhibition rate were also used as the measurement indicators. The strong positive cells were subcloned by limiting dilution method, and this was repeated 3 to 4 times (note that the positive well cells selected in each round need to be expanded and cultured, and then frozen for standby use), until each well of each plate was positive and the titer and inhibition were similar after detection. At this point, the hybridoma cell line was successfully established, and a hybridoma cell line that can stably secrete uniform antibodies was obtained. Single cell clones were picked and those that tested fully positive were transferred to 24-well cell culture plates, 6-well cell culture plates, and 10 cm cell culture dishes for expansion and cryopreservation in time.

[0097] S4. Large-scale preparation of monoclonal antibodies After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are usually prepared in large quantities by in vitro culture and in vivo monoclonal antibody induction. Liquid lysate is injected into the peritoneal cavity of more than ten Balb / c mice over 8 weeks old in advance, with a dose of 0.5 mL / mouse. Hybridoma cells are injected into the peritoneal cavity of mice 1 to 2 weeks later. The state of the mice is observed every day after cell inoculation. In particular, from the 7th day onwards, the abdominal cavity of the mice will swell. Before the death of the mice, ascites is collected aseptically with a disposable syringe. The collected ascites is centrifuged at 12000r / min for 10 minutes to remove the upper fat and lower fibrin, collect the middle layer, and determine its titer and inhibition rate by ELISA. After purification, it is stored at -20°C for standby use to obtain N-acetyl-5-hydroxytryptamine monoclonal antibodies.

[0098] Example 9: N-acetyl-5-hydroxytryptamine ELISA test The titer and specificity of the antiserum were determined by ELISA indirect enzyme-linked immunosorbent assay, and the steps are as follows: The antibody prepared in Example 8 was used to perform an ELISA test for N-acetyl-5-hydroxytryptamine. This test uses competitive immunoassay to determine the N-acetyl-5-hydroxytryptamine content in a liquid sample. The principle is that the N-acetyl-5-hydroxytryptamine in the sample competes with the coupled N-acetyl-5-hydroxytryptamine derivative (HRP-N-acetyl-5-hydroxytryptamine derivative enzyme conjugate) for binding to the limited sites on the antibody coated in the ELISA plate. If there is almost no or no N-acetyl-5-hydroxytryptamine in the liquid sample, the HRP enzyme-coupled N-acetyl-5-hydroxytryptamine derivative will bind to the antibody in the ELISA plate. On the contrary, if the liquid sample contains a large amount or a certain amount of N-acetyl-5-hydroxytryptamine, the enzyme N-acetyl-5-hydroxytryptamine derivative conjugate will reduce the binding with the antibody, thereby weakening the color development signal. Therefore, the absorbance generated by the test is inversely proportional to the N-acetyl-5-hydroxytryptamine content in the liquid sample. The specific steps are as follows:

[0099] Establishment of standard curve for N-acetyl-5-hydroxytryptamine ELISA

[0100] S1. Preparation of Standards

[0101] N-acetyl-5-hydroxytryptamine powder (purchased from Merck) was dissolved in methanol solution to prepare a 1 mg / ml stock solution. The stock solution was diluted with ELISA buffer to 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 standard solutions. The ELISA buffer contained 50.0 mM Tris, 100 mM NaCl and 0.2% BSA.

[0102] S2. Preparation of standard curve using ELISA test method for N-acetyl-5-hydroxytryptamine

[0103] S3. Dilute the anti-N-acetyl-5-hydroxytryptamine antibody prepared in Example 7 with PBS to a final concentration solution of 1:10000, and coat 100 μL / well on a 96-well ELISA plate, and place at 4°C for 12-24h; wash the 96-well ELISA plate coated with the anti-N-acetyl-5-hydroxytryptamine antibody with PBS for 3 times, add 200 μL / well of 0.5% BSA solution, and seal and place at 4°C for 8-16h. Then wash with PBS for 3 times, add 20 μL / well of standard. Then add 100 μL / well of HRP-N-acetyl-5-hydroxytryptamine conjugate at a working concentration; incubate at room temperature for 30 minutes, then wash the plate with PBS 5 times; then add 100 μL of TMB substrate to each well, and incubate at room temperature for 30 minutes. Then add 100 μL of stop solution (2M sulfuric acid) to each well. Measure the absorbance at 450nm. Calibrate the standard curve according to the absorbance value at 450nm corresponding to each standard. The results are shown in the attached figure. Figure 1 shown.

[0104] Example 10: Preparation of 5-HT Alkaline Phosphatase Chemiluminescent Detection Reagent

[0105] Preparation of 5-HT Alkaline Phosphatase Chemiluminescent Detection Reagent Using the Antibody Prepared in Example 8

[0106] S1. A 5-hydroxytryptamine detection kit, in which sample preparation (converting 5-hydroxytryptamine into N-acetyl-5-hydroxytryptamine) is part of the sample dilution, and each sample is incubated with an acetylation reagent to obtain acetylated 5-hydroxytryptamine. This experiment adopts the principle of competitive chemiluminescent immunoassay reagent, and 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. When 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 made with a known standard, and the luminescence value of the unknown sample is calibrated on the standard curve to obtain the concentration of 5-hydroxytryptamine in the unknown sample. It is characterized in that it includes a sample processing solution, N-acetyl-5-hydroxytryptamine labeled with a chemiluminescent marker, and an N-acetyl-5-hydroxytryptamine monoclonal antibody coated on a fixed carrier; The sample processing solution includes an acetylating agent and an organic solvent; The sample treatment solution contains an acetylating agent at a concentration of 0.001 to 100 mg / mL, preferably 20 to 50 mg / mL; the acetylating agent 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, ether and ethyl acetate, preferably ethanol. S2. The chemiluminescent marker in the N-acetyl-5-hydroxytryptamine labeled with the chemiluminescent marker is alkaline phosphatase. S3. The antibody coated on the fixed carrier is an anti-N-acetyl-5-hydroxytryptamine antibody coated on magnetic beads.

[0107] Obtain standard curve: Set the reaction parameters of the alkaline phosphatase chemiluminescence analyzer (see Table 1). The on-machine detection process is: first add sample treatment solution and sample (or standard), incubate at 37℃ for 10 minutes; then add reagent 1 and reagent 2, react at 37℃ for 5 minutes. Add luminescent substrate solution, measure the luminescence value, and the machine automatically calculates the calibration curve and calculates the sample concentration. The calibration curve is as attached. Figure 2 shown.

[0108] Table 1: Alkaline phosphatase chemiluminescence analyzer reaction parameters

[0109]

[0110] The samples were compared by using the 5-HT alkaline phosphatase chemiluminescent detection reagent of the present invention and the 5-HT ELISA detection reagent of a well-known foreign manufacturer. The detection data and data analysis are shown in the attached Figure 3 .

[0111] Example 11: Analog cross-reaction test

[0112] Nine common N-acetyl-5-hydroxytryptamine structural analogs were selected for cross-reaction test and measured using alkaline phosphatase chemiluminescence detection reagent. The nine common N-acetyl-5-hydroxytryptamine structural analogs and their cross-reaction rates are shown in Table 2.

[0113] Table 2: Cross-reactivity results of 9 common N-acetyl-5-hydroxytryptamine structural analogs

[0114] Serotonin Cross-reaction (%) N-acetyl serotonin 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

[0115] Determination results: From the cross-reaction results of the above 9 common N-acetyl-5-hydroxytryptamine structural analogs, it can be seen that the antibody prepared by the present invention has good specificity.

[0116] From the above results, it can be seen that the 5-hydroxytryptamine complete immunogen prepared from the N-acetyl-5-hydroxytryptamine derivative provided by the present invention has strong immunogenicity, the generated antibody has high specificity, and has good affinity with N-acetyl-5-hydroxytryptamine. The alkaline phosphatase chemiluminescent reagent prepared using the above antibody has good stability and high sensitivity, and can realize high-throughput and rapid detection of serotonin on a fully automatic chemiluminescence analyzer, and has the advantages of simple operation, high sensitivity, strong specificity, accurate results, etc., and can also effectively reduce the cost of N-acetyl-5-hydroxytryptamine detection, which is conducive to clinical promotion and use.

[0117] 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 structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in 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 formula (I): Wherein, R is a connecting group, R is -(CH2) n —COOH, n is an integer between 1 and 10.

2. An N-acetyl-5-hydroxytryptamine immunogen, characterized in that It has the structural formula shown in formula (II): Where R is —(CH2) n —COOH, n is an integer between 1 and 10, and the carrier is an immunogenic protein or polypeptide; preferably, R is —(CH2) n —COOH, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or poly-lysine.

3. An anti-N-acetyl-5-hydroxytryptamine specific antibody produced by immunizing an animal with an immunogen, characterized in that: The anti-N-acetyl-5-hydroxytryptamine specific antibody is obtained by immunizing an animal with the N-acetyl-5-hydroxytryptamine immunogen according to claim 2.

4. The antibody according to claim 3, characterized in that The antibody is a complete protein molecule or a polypeptide fragment having the ability to specifically bind 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 is 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 comprises: preparing the N-acetyl-5-hydroxytryptamine derivative according to claim 1; and connecting the N-acetyl-5-hydroxytryptamine derivative with a carrier to obtain the N-acetyl-5-hydroxytryptamine immunogen; wherein the carrier is a protein or polypeptide with immunogenicity; The steps of preparing the N-acetyl-5-hydroxytryptamine derivative include: S1, subjecting N-acetyl-5-hydroxytryptamine 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 ester 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 hours to obtain the N-acetyl-5-hydroxytryptamine derivative.

8. The preparation method according to claim 7, characterized in that: The step S1 comprises the following steps: A1. Dissolving N-acetyl-5-hydroxytryptamine and halo-(CH2)n-COO-tert-butyl ester in di-N,N-dimethylformamide (DMF) to obtain a mixed solution; wherein the molar ratio of the N-acetyl-5-hydroxytryptamine to the halo-(CH2)n-COO-tert-butyl ester is 0.8-1.6:1; preferably, after obtaining the mixed solution, the method further comprises heating the mixed solution to a constant temperature, more preferably, the temperature of heating to the constant temperature is 70-100° C., and the constant temperature time is greater than or equal to 32 hours; A2, adding ethyl acrylate to the mixed solution to obtain a reaction mixture, and stirring and mixing the reaction mixture at a low temperature of 0 to 8° C. to obtain a solid-liquid mixture; A3. Filter, wash, purify and vacuum dry the reaction mixture 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 S2 of the carrier and the N-acetyl-5-hydroxytryptamine derivative comprises: B1, preparing a carrier solution and an N-acetyl-5-hydroxytryptamine derivative solution; preferably, the carrier is serum protein, keyhole limpet hemocyanin, thyroglobulin or poly-lysine; B2, adding 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, stirring the mixed solution at 2-10° C. overnight (or reacting at room temperature for 2 hours) to obtain the crude N-acetyl-5-hydroxytryptamine immunogen; B4. Purify the crude N-acetyl-5-hydroxytryptamine immunogen to obtain the pure N-acetyl-5-hydroxytryptamine immunogen, preferably by dialysis bag dialysis.

11. The preparation method according to claim 7, characterized in that: In the step B1, the step of preparing the carrier solution includes dissolving the carrier in a phosphate buffer solution of 0.05 to 0.2 M and a pH of 8.0 to 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-hydroxysulfosuccinimide (Sulfo-NHS) in N,N-dimethylformamide, methanol and 5 to 20 mM, pH 4.0 to 6.0 phosphate buffer and stirring at room temperature 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 to 8:

1.

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

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