Preparation method of key antigen marker for dehydroepiandrosterone sulfate chemiluminescence detection

Abstract

The invention relates to a preparation method of a key antigen marker for dehydroepiandrosterone sulfate chemiluminescence detection and application of the key antigen marker in a detection reagent. Dehydroepiandrosterone which is easy to obtain, low in price and similar to dehydroepiandrosterone sulfate (DHEA-S) in chemical structure is selected as a starting material of a competitive antigen, an optimal synthesis process route is designed according to a designed target product by considering a cost factor, separation and purification difficulty and a later amplification production factor, and the dehydroepiandrosterone is synthesized through a simple reaction. The preparation method comprises the following steps: preparing an epiandrosterone derivative, directly coupling the epiandrosterone derivative with acridine salt, and purifying to obtain the epiandrosterone derivative which can be used as a key competitive antigen raw material of a DHEA-S magnetic particle chemiluminescence immunoassay reagent. The detection reagent and the raw materials are easy to obtain, the preparation is simple, the specificity is good, the result is accurate, the cost is low, and the scale detection of the dehydroepiandrosterone sulfate is met.

Description

Technical Field

[0001] The present application relates to a preparation method and application of a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate, belonging to the field of chemiluminescent immunoassay. Background Art

[0002] Dehydroepiandrosterone sulfate (DHEA-S) is an important steroid hormone secreted by the zona reticularis of the adrenal cortex. It is present in high concentrations in the blood and circulates primarily as a sulfate ester. DHEA-S is converted to dehydroepiandrosterone (DHEA) in the body through desulfurization and further metabolized into biologically active androgens or estrogens. Compared with other androgens (such as testosterone), DHEA-S has a longer half-life (approximately 8-10 hours) and less volatile serum levels. Therefore, it is widely used as an important biomarker for assessing adrenal function, sex hormone disorders, and related diseases.

[0003] The measurement of serum DHEA-S concentration is commonly used clinically to assist in the diagnosis of conditions such as benign adrenal tumors, adrenal cancer, and adrenal hyperplasia (congenital or adult-onset). It can also be used to differentiate between ovarian and adrenal sources of androgen excess. Furthermore, DHEA-S levels are also crucial for the diagnosis of conditions such as polycystic ovary syndrome (PCOS), hirsutism, virilization, and precocious puberty in children.

[0004] Currently, commercially available DHEA-S detection reagents primarily utilize the competitive chemiluminescent immunoassay (CLIA) method. Because DHEA-S is a small molecule hapten, its structure is not suitable for sandwich assays, so the competitive assay principle is commonly used. This method relies on a competing antigen—a marker that binds to DHEA-S-specific antibodies and simultaneously provides a luminescent signal. The structure of the competing antigen directly determines the sensitivity, specificity, and stability of the assay.

[0005] Furthermore, in actual industrial production, preparation cost, product separation and purification efficiency, yield, and process scale-up stability are key indicators for evaluating the quality of a synthetic process. In another patent application filed on the same day, the applicant disclosed the structure and preparation method of an acridinium-labeled epiandrosterone derivative, a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate. While this preparation method also produces the desired target product, the expensive raw material, acridinium salt, undergoes three steps of reaction to obtain the target product, resulting in a larger amount of material used, more complex separation and purification, a longer production cycle, and higher costs. The method involves first preparing an epiandrosterone derivative intermediate with a carboxylic acid group at the molecular end. Using the carboxylic acid of an acridinium salt as a starting material, a Boc-protected aminoacridinium salt is prepared. Due to the high polarity of both the acridinium salt carboxylic acid and the Boc-protected aminoacridinium salt product, this step of separation and purification is difficult. Deprotection is then performed to obtain a terminally amino-modified acridinium salt intermediate. The epiandrosterone derivative intermediate with a carboxylic acid group at the molecular end is then directly coupled with the terminally amino-modified acridinium salt intermediate. Due to the high polarity of both the terminally amino-modified acridinium salt of the starting material and the product, this step of separation and purification is difficult. After purification, the target molecule is obtained. In this synthetic route, the expensive acridinium salt undergoes three reaction steps to obtain the target product. Furthermore, the polarity of the acridinium salt raw material and the products of each step is relatively high, resulting in poor separation, long separation cycles, and high losses, leading to high synthesis costs.

[0006] While this method has achieved the synthesis of the target molecule, it still faces challenges in scale-up production or specific industrial applications, including high raw material costs, cumbersome separation and purification steps, a need to improve the overall yield of the target product, and long production cycles. Therefore, the present invention, building on the aforementioned method, further optimizes the process flow to achieve lower costs, higher yields, and more efficient purification, making it more suitable for large-scale production and industrialization. Summary of the Invention

[0007] Problems to be solved by the invention To address the shortcomings and deficiencies of the above-mentioned prior art, the present invention provides a method for preparing a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate (DHEA-S) and discloses its use in preparing a DHEA-S magnetic microparticle chemiluminescent immunoassay reagent or a DHEA-S magnetic microparticle chemiluminescent immunoassay kit. The preparation method of the present invention uses dehydroepiandrosterone (DHEA-S), a readily available and inexpensive raw material with a chemical structure similar to dehydroepiandrosterone sulfate (DHEA-S), as the starting material for the competitive antigen. Based on the designed target product, an optimal synthesis route is designed taking into account cost factors, isolation and purification difficulties, and subsequent scale-up production factors. Through a simple reaction, an epiandrosterone derivative is prepared, which is then directly coupled with an acridinium salt. After purification, the epiandrosterone derivative is obtained, which can be used as the key competitive antigen raw material for the DHEA-S magnetic microparticle chemiluminescent immunoassay reagent.

[0008] The detection reagent of the present invention has readily available raw materials, is simple to prepare, has good specificity, produces accurate results, and is low in cost, and can meet the requirements for large-scale detection of dehydroepiandrosterone sulfate.

[0009] Solutions for solving problems The present invention provides a method for preparing a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate, wherein the key antigen marker has a structural formula shown in Formula 1: (Formula 1) The preparation method comprises the following steps: Step a: providing dehydroepiandrosterone and succinic anhydride, and subjecting the dehydroepiandrosterone and succinic anhydride to an esterification reaction in the presence of a DMF solvent and a catalytic amount of a DMAP catalyst; and obtaining an intermediate represented by Formula 2 after post-treatment; (Formula 2) Step b: The intermediate of Formula 2 is reacted with Boc-ethylenediamine in the presence of DMF solvent, activator HBTU and a base to undergo an amide condensation reaction, and the post-treated reaction product is subjected to a deprotection treatment to remove the Boc group in the reaction product to obtain the ethylenediamine-dehydroepiandrosterone derivative of Formula 3; (Formula 3) Step c: An ethylenediamine-dehydroepiandrosterone derivative represented by Formula 3 and an acridinium salt derivative having a carboxylic acid group represented by Formula 4 are subjected to an amide condensation reaction in the presence of a DMF solvent, an activating agent HBTU, and a base. After post-treatment and purification, a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate represented by Formula 1 is obtained;

[0010] (Formula 4).

[0011] Furthermore, in step a, the post-treatment includes removing DMF by distillation under reduced pressure and beating with dilute hydrochloric acid, and filtering and drying to obtain an off-white solid product.

[0012] Furthermore, in step b, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding an appropriate amount of water, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate, and purification by column to obtain the product; the deprotection treatment includes: adding the product collected by the column to a dioxane solution of HCl, and reacting after deprotection to obtain the ethylenediamine-dehydroepiandrosterone derivative shown in Formula 3.

[0013] Furthermore, in step c, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding DCM, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate and purification by column to obtain the product.

[0014] The present invention also provides a key antigen marker prepared by any of the preparation methods described above.

[0015] The present invention also provides the use of the key antigen marker in the preparation of a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay reagent or a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay kit.

[0016] The present invention also discloses a chemiluminescent immunoassay reagent, comprising: the key antigen marker mentioned above; a biotin marker for dehydroepiandrosterone sulfate (DHEA-S) antibody; streptavidin magnetic beads and a working calibrator.

[0017] Effects of the Invention 1. The present invention uses dehydroepiandrosterone as the starting material. Dehydroepiandrosterone is highly similar in structure to the target detection substance dehydroepiandrosterone sulfate (DHEA-S), is readily available on the market, and has a cost far lower than readily available DHEA-S derivatives on the market. This significantly reduces the synthesis cost of the competing antigen and facilitates large-scale industrial preparation.

[0018] 2. The present invention uses dehydroepiandrosterone as the starting material, which has a chemical structure similar to but not identical to DHEA-S. It exhibits moderate affinity when binding to antibodies, which is weaker than the DHEA-S antigen in the sample, ensuring detection sensitivity, but stronger than non-specific antigens, improving the accuracy of the competitive reaction, thereby achieving the construction of a good immune competition system.

[0019] 3. In the synthesis steps of the present invention, dehydroepiandrosterone (DHEA), which is inexpensive and less polar (relative to the polarity of the acridinium salt), is used as the starting material. The synthesis proceeds through a carboxyl derivative of DHEA (Formula 2), a Boc-protected amino derivative, and an amino derivative (Formula 3). The carboxyl derivative of DHEA (Formula 2) and the Boc-protected amino derivative have lower polarity (relative to the polarity of the acridinium salt) and are easily separated and purified on a silica gel column. The amino derivative (Formula 3) undergoes a substantially quantitative reaction, thus requiring no purification. These steps provide high yields and facilitate separation and purification, improving efficiency and reducing costs. Finally, the intermediate DHEA derivative with a terminal amino group is reacted with the acridinium salt carboxylic acid to obtain the target product. In this synthetic route, the expensive acridinium salt raw material is reacted in a single step to obtain the target product, reducing the amount used and the cost. This offers significant cost advantages during scale-up production.

[0020] 4. The present invention chemically synthesizes epiandrosterone derivatives to produce single-molecule chemiluminescent labels with a 1:1 ratio and high yield. Using these epiandrosterone derivatives to detect the content of dehydroepiandrosterone sulfate in clinical samples avoids the problem of being unable to precisely control the labeled amounts of dehydroepiandrosterone sulfate and the luminescent molecule acridinium salt, thereby significantly reducing batch variability and improving the accuracy of clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , Preparation Flowchart of Example 1 Figure 2 , Mass spectrometry test data diagram of compound 2 shown in formula 2 Figure 3 , Mass spectrometry data of compound 4 Figure 4 , Mass spectrometry test data of compound 5 shown in formula 3 Figure 5 , Mass spectrometry test data of compound 7 shown in formula 1 Figure 6 , the test result diagram shown in the examples of this application. DETAILED DESCRIPTION

[0022] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0023] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0025] It will be understood that as used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0026] References in this specification to "one or more specific / preferred embodiments / solutions," "another or other specific / preferred embodiments / solutions," "one or another embodiment / solution," "one or another technical solution," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) associated with the embodiment described are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any appropriate manner in various embodiments.

[0027] The term "comprises" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0028] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0029] In the present invention, the solvent used in the reaction system is a solvent or a mixed solvent that can dissolve the raw materials to form a homogeneous solution and does not react with the raw materials, such as DMSO, DCM, DMF, etc.

[0030] As summarized above, the present application discloses a method for preparing a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate, wherein the key antigen marker has the structural formula shown in Formula 1: (Formula 1) The preparation method comprises the following steps: Step a: providing dehydroepiandrosterone and succinic anhydride, and subjecting the dehydroepiandrosterone and succinic anhydride to an esterification reaction in the presence of a DMF solvent and a catalytic amount of DMAP (4-dimethylaminopyridine) catalyst; and post-processing to obtain an intermediate represented by Formula 2; The present invention uses dehydroepiandrosterone as the starting material. Dehydroepiandrosterone is highly similar in structure to the target detection substance dehydroepiandrosterone sulfate (DHEA-S), is readily available on the market, and has a cost far lower than readily available DHEA-S derivatives on the market. This significantly reduces the synthesis cost of the competing antigen and facilitates large-scale industrial preparation.

[0031] Step b: The intermediate of Formula 2 is reacted with Boc-ethylenediamine in the presence of DMF solvent, an activating agent HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), a coupling agent commonly used in solid-phase peptide synthesis, and a base to undergo an amide condensation reaction. The post-treated reaction product is then deprotected to remove the Boc group therein, thereby obtaining the ethylenediamine-dehydroepiandrosterone derivative of Formula 3. The epiandrosterone derivative provided by the present invention has specific flexibility and spatial conformation by introducing succinic anhydride and ethylenediamine as spacer arms. It can be coupled with acridinium ester chemiluminescent markers with excellent coupling efficiency while maintaining antigen recognition ability, thereby constructing a DHEA-S competitive antigen with stable performance, high luminescence sensitivity and a clear synthetic route.

[0032] Step c: An ethylenediamine-dehydroepiandrosterone derivative shown in Formula 3 and an acridinium salt derivative with a carboxylic acid group shown in Formula 4 are subjected to an amide condensation reaction in the presence of a DMF solvent, an activating agent HBTU, and a base. After post-treatment and purification, a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate shown in Formula 1 is obtained.

[0033] Furthermore, in step a, the post-treatment includes removing DMF by distillation under reduced pressure and beating with dilute hydrochloric acid, and filtering and drying to obtain an off-white solid product.

[0034] Furthermore, in step b, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding an appropriate amount of water, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate, and purification by column to obtain the product; the deprotection treatment includes: adding the product collected by the column to a dioxane solution of HCl, and reacting after deprotection to obtain the ethylenediamine-dehydroepiandrosterone derivative shown in Formula 3.

[0035] In the above-mentioned synthetic steps a and b, dehydroepiandrosterone, which is cheap and has a lower polarity (relative to the polarity of the acridinium salt), is selected as the starting material. The synthesis involves a carboxyl derivative of dehydroepiandrosterone (Formula 2), a Boc-protected amino derivative, and an amino derivative (Formula 3). The carboxyl derivative of dehydroepiandrosterone (Formula 2) and the Boc-protected amino derivative have lower polarity and are easily separated and purified by silica gel column, while the amino derivative (Formula 3) undergoes a substantially quantitative reaction and therefore does not require purification. These steps have good reaction yields and are convenient for separation and purification, thereby improving efficiency and reducing costs.

[0036] Furthermore, in step c, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding DCM, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate and purification by column to obtain the product.

[0037] In the synthetic route of the present invention, the expensive acridinium salt raw material undergoes a single reaction step to yield the target product, reducing the required dosage and significantly lowering industrial production costs. The other raw material, dehydroepiandrosterone, is inexpensive, and the products after each reaction step are relatively low in polarity, making separation easier, shortening the separation cycle, and minimizing losses, significantly reducing synthesis costs.

[0038] The present invention also provides a key antigen marker prepared by any of the preparation methods described above.

[0039] The present invention also provides the use of the key antigen marker in the preparation of a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay reagent or a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay kit.

[0040] The present invention also discloses a chemiluminescent immunoassay reagent, comprising: the key antigen marker mentioned above; a biotin marker for DHEA-S antibodies; streptavidin magnetic beads and a working calibrant.

[0041] Examples With reference to the above implementation content, in order to make the technical solution of the present application more specific, clear and easy to understand, the technical solution of the present application is now given as an example. However, it should be noted that the following embodiments are listed to illustrate the present invention. Those skilled in the art can understand that the example is only an illustrative description and not an exhaustive description.

[0042] Example 1 See Figure 1 The preparation process of this embodiment is shown in the figure, specifically: Step 1: Dehydroepiandrosterone (Compound 1) (1 g, 1 eq) and succinic anhydride (1.4 g, 4 eq) were added to DMF (20 ml). Catalyst DMAP (0.43 g, 1 eq) was added and the mixture was heated at 50°C overnight. The solvent was evaporated and dilute hydrochloric acid was added to precipitate a solid. The solid was filtered, washed with water, and dried to obtain a dehydroepiandrosterone carboxylic acid derivative (Compound 2) in an 85% yield. Mass spectrometry results: 386.95 (M-1) and 775.70 (2M-1), see Figure 2 .

[0043] Step 2: Compound 2 (0.5 g, 1 eq) and Boc-ethylenediamine (compound 3) (0.3 g, 1.5 eq) were added to DMF (10 ml), followed by the addition of HBTU (0.72 g, 1.5 eq). Under nitrogen protection, DIEA (0.5 g, 3 eq) was added dropwise in an ice-water bath, and the mixture was allowed to react overnight at room temperature. After the reaction was complete, DMF was removed under reduced pressure, an appropriate amount of water was added, and the mixture was washed twice with a small amount of dilute HCl, washed with saturated brine, washed with saturated NaHCO3 water, and washed with saturated brine water. The mixture was dried over anhydrous sodium sulfate and purified by simple column chromatography. The eluent used was PE:EA = 2:1-1:1 to obtain compound 4 with a yield of 90%. The mass spectrum result was 531.55 (M+1), see Figure 3 .

[0044] Step 3: Compound 4 (0.2 g) was added to a solution of HCl in dioxane (10 ml). After dissolving, the mixture was reacted at room temperature for 5 h. A white solid was formed. The solvent was evaporated to obtain compound 5, which was used directly in the next step without purification. Mass spectrum: 431.80 (M+1), see Figure 4 .

[0045] Step 4: Compounds 5 and 6 (0.23 g, 1 eq) were added to anhydrous DMF (10 ml), followed by HBTU (0.225 g, 1.5 eq). Under nitrogen protection, DIEA (0.153 g, 3 eq) was then added dropwise in an ice-water bath. The reaction was allowed to proceed overnight at room temperature. After the reaction, the solution was green. DMF was removed under reduced pressure, and DCM was added. The solution was washed with dilute HCl, saturated brine, saturated NaHCO3, and saturated brine. The solution was purified by column chromatography using a 15:1 ratio of DCM to methanol as the eluent to obtain a single-molecule chemiluminescent acridinium salt-labeled epiandrosterone derivative (Compound 7). Mass spectrum: 995.01649 (M-1), see Figure 5 .

[0046] It should be understood that in the preparation process of the embodiments of the present application, the specific reaction conditions, including but not limited to: reaction temperature, time, pressure, pH, etc. can be adaptively adjusted according to actual conditions, and the relevant conditions and parameters disclosed in the embodiments of the present application do not mean to limit the present application.

[0047] It should be understood that the purification, drying, extraction and other processes used in the embodiments of the present application can be carried out in other ways known to those skilled in the art.

[0048] Example 2: Preparation method of biotin label for DHEA-S antibody: Dissolve biotin-NHS active ester in DMSO to a concentration of 4 mg / ml. Prepare a 1 mg / ml solution of DHEA-S antibody in 0.1 M PB buffer (pH 7.4). Based on the desired labeling ratio of biotin-NHS active ester to DHEA-S antibody, add an appropriate amount of the biotin-NHS active ester DMSO solution to the DHEA-S antibody PB solution. Shake and react at room temperature for 1.5 hours. Then, dialyze and purify three times in 0.1 M PB (pH 7.4) to obtain the biotin-labeled DHEA-S antibody.

[0049] Working calibrator target values: S0 (0 μg / dL), S1 (4.5 μg / dL), S2 (15 μg / dL), S3 (45 μg / dL), S4 (450 μg / dL), S5 (980 μg / dL).

[0050] Streptavidin magnetic beads: Dilute the streptavidin magnetic beads with magnetic bead buffer.

[0051] Reagent R1: Dilute the biotinylated label of the DHEA-S antibody in reagent buffer.

[0052] Reagent R2: Dissolve compound 7 in DMSO and then dilute compound 7 with reagent buffer.

[0053] The results of the calibration test using a chemiluminescence analyzer are shown in the following table. Figure 6 The results showed a good correlation between concentration and luminescence value. As the concentration of DHEA sulfate increased, the luminescence value gradually decreased, showing an inverse relationship. Therefore, the detection product of this application can accurately detect the content of DHEA sulfate and has great application prospects in the quantitative detection of DHEA sulfate in clinical samples.

[0054] Table 1 Test data Calibrators Luminescence value (RLU) S0 1338980 S1 1107993 S2 960089 S3 789451 S4 584735 S5 518799

Claims

1. A method for preparing a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate, characterized in that: The key antigen marker has the structural formula shown in Formula 1: (Equation 1) The preparation method comprises the following steps: Step a: providing dehydroepiandrosterone and succinic anhydride, and subjecting the dehydroepiandrosterone and succinic anhydride to an esterification reaction in the presence of a DMF solvent and a catalytic amount of a DMAP catalyst; and obtaining an intermediate represented by Formula 2 after post-treatment; (Formula 2) Step b: The intermediate of Formula 2 is reacted with Boc-ethylenediamine in the presence of DMF solvent, activator HBTU and a base to undergo an amide condensation reaction, and the post-treated reaction product is subjected to a deprotection treatment to remove the Boc group in the reaction product to obtain the ethylenediamine-dehydroepiandrosterone derivative of Formula 3; (Formula 3) Step c: An ethylenediamine-dehydroepiandrosterone derivative shown in Formula 3 and an acridinium salt derivative with a carboxylic acid group shown in Formula 4 are subjected to an amide condensation reaction in the presence of a DMF solvent, an activating agent HBTU, and a base. After post-treatment and purification, a key antigen marker for chemiluminescent detection of dehydroepiandrosterone sulfate shown in Formula 1 is obtained. (Formula 4).

2. The method for preparing the key antigen marker according to claim 1, characterized in that: In step a, the post-treatment includes removing DMF by distillation under reduced pressure and beating with dilute hydrochloric acid, and filtering and drying to obtain an off-white solid product.

3. The method for preparing the key antigen marker according to claim 1, characterized in that: In step b, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding an appropriate amount of water, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate, and purification by column to obtain the product; the deprotection treatment includes adding the product collected by the column to a dioxane solution of HCl, and reacting after deprotection to obtain the ethylenediamine-dehydroepiandrosterone derivative shown in Formula 3.

4. The method for preparing the key antigen marker according to claim 1, characterized in that: In step c, the base is N,N-diisopropylethylamine (DIEA), and the post-treatment includes removing DMF by distillation under reduced pressure, adding DCM, and washing with one or more of dilute hydrochloric acid, saturated saline, and saturated NaHCO3, followed by drying with anhydrous sodium sulfate and purification by column to obtain the product.

5. The key antigen marker obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the key antigen marker according to claim 5 in the preparation of a dehydroepiandrosterone sulfate magnetic particle chemiluminescent immunoassay reagent or a dehydroepiandrosterone sulfate magnetic particle chemiluminescent immunoassay kit.

7. A chemiluminescent immunoassay reagent, characterized in that: include: The key antigen marker according to claim 5; Biotin labeling of dehydroepiandrosterone sulfate (DHEA-S) antibodies; Streptavidin magnetic beads; Working calibrator.

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