Dehydroepiandrosterone carboxylic acid NHS active ester marker as well as preparation method and application thereof

Abstract

The invention relates to a dehydroepiandrosterone carboxylic acid NHS active ester marker as well as a preparation method and application thereof, dehydroepiandrosterone is selected as an initial raw material of a competitive antigen, the source is wide, the cost is low, dehydroepiandrosterone carboxylic acid NHS active ester is prepared through a two-step reaction, the dehydroepiandrosterone carboxylic acid NHS active ester can be used as an active intermediate, and the dehydroepiandrosterone carboxylic acid NHS active ester marker can be used as a competitive antigen. As a marker for binding protein or other molecules; for example, the dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay reagent with high sensitivity and good specificity can be prepared at low cost.

Description

Technical Field

[0001] The present application relates to a dehydroepiandrosterone carboxylic acid NHS active ester label, a preparation method thereof, and an application thereof, and belongs to the field of chemiluminescence immunoassay. Background Art

[0002] Chemiluminescence immunoassay (CLIA) is a detection and analysis technique that combines a chemiluminescence determination technique with high sensitivity and an immune reaction with high specificity, and is used for the detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. It is the latest immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay.

[0003] Dehydroepiandrosterone sulfate (DHEA-S) belongs to a small molecule hapten. In chemiluminescence immunoassay, the competitive method is generally used for detection. The competitive antigen in the detection reagent is a key raw material. The affinity between this competitive antigen and the antibody should not be too large or too small. If it is too large, it is difficult for the dehydroepiandrosterone sulfate (DHEA-S) antigen in the sample to bind to the antibody. If it is too small, it cannot bind well to the antibody, affecting the detection effect.

[0004] Dehydroepiandrosterone sulfate (DHEA-S) is a steroid hormone mainly synthesized by the adrenal gland. In these tissues containing sulfatase activity, dehydroepiandrosterone sulfate can be converted into non-steroidal dehydroepiandrosterone (DHEA). Therefore, dehydroepiandrosterone and / or dehydroepiandrosterone sulfate may be partially metabolized into active androgens and estrogens. The level of dehydroepiandrosterone sulfate in serum and plasma is the highest among all steroids. After the level of dehydroepiandrosterone sulfate reaches the maximum level at around thirty years old, for both men and women, with the increase of age, the level of dehydroepiandrosterone sulfate decreases. The half-life of dehydroepiandrosterone sulfate is 8 to 10 hours. Compared with the half-life of 30 to 60 minutes of other androgens, the long half-life of dehydroepiandrosterone sulfate in serum combined with the limited daily changes makes dehydroepiandrosterone sulfate a convenient marker for evaluating adrenal products. Most of the dehydroepiandrosterone (DHEA) in serum exists in the form of sulfate conjugates (DHEA-S). Approximately 90% of the DHEA-S in the blood circulation comes from the zona reticularis of the adrenal cortex. The concentration in serum is mostly used to evaluate the situation of suspected excessive secretion of adrenal androgens.

[0005] The detection of DHEA-S helps to diagnose adrenal benign tumors (benign tumors located in the adrenal cortex), adrenal malignant tumors, and adrenal hyperplasia (which can be hereditary or primary in adults), as well as to distinguish similar symptoms caused by ovarian benign and malignant tumors.

[0006] DHEA-S concentration is usually measured simultaneously with FSH, LH, calcitonin, estrogen and testosterone to assist in the diagnosis of polycystic ovary syndrome (PCOS) and rule out other causes of infertility, amenorrhea and hirsutism. DHEA-S testing can be used to study and analyze the causes of virilization in girls and precocious puberty in boys in combination with other hormones. DHEA-S testing can be used for the differential diagnosis of hirsutism (abnormal hair growth, in female patients with beard, increased levels of dehydroepiandrosterone sulfate may be associated with virilizing adrenal tumors), virilization (masculinization of women), polycystic ovary syndrome, adrenal tumors and Cushing's syndrome.

[0007] At present, there are few chemiluminescence immunoassay reagents for dehydroepiandrosterone sulfate magnetic particles in the domestic market, mainly imported reagents, and the testing cost is high. Summary of the Invention

[0008] The purpose of this application is to provide a dehydroepiandrosterone carboxylic acid NHS active ester label, its preparation method and application, which can prepare a chemiluminescence immunoassay reagent for dehydroepiandrosterone sulfate magnetic particles with high sensitivity and good specificity at low cost.

[0009] To achieve the above object, this application provides the following technical solutions:

[0010] In the first aspect, this application provides a dehydroepiandrosterone carboxylic acid NHS active ester label, which has the structural formula shown in Formula I:

[0011] (Formula I)

[0012]

[0013] In the second aspect, this application provides a preparation method of a dehydroepiandrosterone carboxylic acid NHS active ester label, which includes the following steps:

[0014] S1. Provide dehydroepiandrosterone and succinic anhydride, add the dehydroepiandrosterone and succinic anhydride to an organic solvent, add a catalyst and then heat the reaction and remove impurities to obtain a dehydroepiandrosterone carboxylic acid derivative;

[0015] S2. Add the dehydroepiandrosterone carboxylic acid derivative and N-hydroxysuccinimide (N-Hydroxysuccinimide, HOSu / NHS) to an organic solvent, add a cross-linking agent and then react and remove impurities to obtain the dehydroepiandrosterone carboxylic acid NHS active ester.

[0016] In one or more feasible embodiments, in step S1, the catalyst is 4-dimethylaminopyridine (4-Dimethylaminopyridine, DMAP).

[0017] In one or more feasible embodiments, heating the reaction and removing impurities after adding the catalyst includes:

[0018] Add the catalyst DMAP, heat to 45 - 55 °C and react overnight, then distill off the solvent, add dilute hydrochloric acid to precipitate the solid, and then filter, wash with water and dry in sequence.

[0019] In one or more feasible embodiments, in step S2, the cross-linking agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride, EDC.HCl).

[0020] In one or more feasible embodiments, reacting and removing impurities after adding the cross-linking agent includes:

[0021] Add EDC.HCl, react at room temperature for more than 3 h; then distill off the solvent, perform extraction and drying, and then purify by column chromatography.

[0022] In one or more feasible embodiments, the organic solvent is selected from any one or more of N,N-dimethylformamide (N,N-Dimethylformamide, DMF), dichloromethane (Dichloromethane, DCM), ethyl acetate or dimethyl sulfoxide (Dimethyl sulfoxide, DMSO).

[0023] In a third aspect, the present application provides a dehydroepiandrosterone carboxylic acid NHS active ester label prepared by the preparation method described in the second aspect.

[0024] In a fourth aspect, the present application provides an application of the dehydroepiandrosterone carboxylic acid NHS active ester label described in the first aspect or the third aspect in the preparation of a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay reagent or a dehydroepiandrosterone sulfate magnetic particle chemiluminescence immunoassay kit.

[0025] In a fifth aspect, the present application provides a preparation method of a chemiluminescence immunoassay reagent, which includes:

[0026] Provide the dehydroepiandrosterone carboxylic acid NHS active ester label described in the third aspect;

[0027] Use the dehydroepiandrosterone carboxylic acid NHS active ester label and AE acridinium ester (acridinium ester, AE(NSP-SA-NHS)) to label bovine serum albumin (Bovine albumin, BSA) to obtain a DHEA-BSA-AE label.

[0028] In one or more feasible embodiments, the labeling of BSA with the dehydroepiandrosterone carboxylic acid NHS active ester label and AE(NSP-SA-NHS) includes:

[0029] Adding the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester to the PB solution of BSA, and purifying after reaction to obtain the DHEA-BSA label;

[0030] Adding the DMF solution of the AE(NSP-SA-NHS) to the PB solution of the DHEA-BSA label, and purifying after reaction to obtain the DHEA-BSA-AE label.

[0031] In one or more feasible embodiments, before adding the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester to the PB solution of BSA, it further includes:

[0032] Dissolving the dehydroepiandrosterone carboxylic acid NHS active ester in DMF to prepare a 2 mg / ml solution;

[0033] Dissolving the AE(NSP-SA-NHS) in DMF to prepare a 4 mg / ml solution;

[0034] Dissolving BSA in 0.1M PB pH 7.4 to prepare a 1 mg / ml solution.

[0035] In one or more feasible embodiments, adding the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester to the PB solution of BSA, and purifying after reaction to obtain the DHEA-BSA label, includes:

[0036] According to the labeling ratio of the dehydroepiandrosterone carboxylic acid NHS active ester to BSA, taking an appropriate amount of the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester and adding it to the PB solution of BSA, shaking and reacting at room temperature for 1.5 h, and then dialyzing and purifying 3 times with 0.1M PB pH 7.4 to obtain the DHEA-BSA label.

[0037] In one or more feasible embodiments, adding the DMF solution of the AE(NSP-SA-NHS) to the PB solution of the DHEA-BSA label, and purifying after reaction to obtain the DHEA-BSA-AE label, includes:

[0038] According to the labeling ratio of AE (NSP-SA-NHS) and DHEA-BSA, an appropriate amount of the DMF solution of AE was taken and added to the PB buffer solution of DHEA-BSA, and the mixture was shaken and reacted for 1.5 h. Then, it was dialyzed and purified three times with 0.1 M PB pH 6.8 to obtain the DHEA-BSA-AE labeled product, which is the key competitive antigen raw material for the chemiluminescent immunoassay reagent of dehydroepiandrosterone magnetic particles.

[0039] In a sixth aspect, the present application provides a DHEA-BSA-AE labeled product prepared by the preparation method of the chemiluminescent immunoassay reagent according to the fifth aspect.

[0040] Through the embodiments of the present application, the beneficial effects of the present application are as follows:

[0041] The present application selects dehydroepiandrosterone as the starting material for the competitive antigen, which has a wide source and low cost. Then, dehydroepiandrosterone carboxylic acid NHS active ester is prepared through two-step reactions and can be used as an active intermediate and a labeling agent for binding to proteins or other molecules.

[0042] Subsequently, it is coupled with the carrier protein BSA, and the dehydroepiandrosterone derivative can be covalently linked to BSA. After purification, it is labeled with acridinesulfonamide, and the key competitive antigen raw material for the chemiluminescent immunoassay reagent of dehydroepiandrosterone magnetic particles is obtained after purification.

[0043] The above description is only an overview of the technical solution of the present application. In order to understand the technical means of the present application more clearly and implement it according to the content of the specification, the following describes in detail with the preferred embodiments of the present application in conjunction with the accompanying drawings. Description of the Drawings

[0044] Figure 1 It is the mass spectrum of the dehydroepiandrosterone carboxylic acid derivative shown in one of the embodiments of the present application;

[0045] Figure 2 It is the mass spectrum of the dehydroepiandrosterone carboxylic acid NHS active ester shown in one of the embodiments of the present application;

[0046] Figure 3 It is the sample correlation R shown in one of the embodiments of the present application 2 data graph. Detailed Embodiments

[0047] The following describes the implementation modes of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. The present application can also be implemented or applied through other different specific implementation modes, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0048] It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present application in a schematic manner. Therefore, only the components related to the present application are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The type, quantity, and ratio of each component in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0049] Some exemplary embodiments of the present application are described for the purpose of illustration. It should be understood that the present application can be implemented in other ways not specifically shown in the drawings.

[0050] In at least one embodiment of the present application, a dehydroepiandrosterone carboxylic acid NHS active ester marker is provided, which has the structural formula shown in Formula I:

[0051] (Formula I)

[0052]

[0053] In at least one embodiment of the present application, a preparation method of a dehydroepiandrosterone carboxylic acid NHS active ester marker is also provided, which includes the following steps:

[0054] S1. Provide dehydroepiandrosterone and succinic anhydride, add the dehydroepiandrosterone and succinic anhydride into an organic solvent, add a catalyst, heat the reaction and remove impurities to obtain a dehydroepiandrosterone carboxylic acid derivative;

[0055] S2. Add the dehydroepiandrosterone carboxylic acid derivative and HOSu into an organic solvent, add a crosslinking agent, react and remove impurities to obtain the dehydroepiandrosterone carboxylic acid NHS active ester.

[0056] Specifically, as follows:

[0057]

[0058] By way of example and not limitation, in step S1, the catalyst is DMAP; the heating reaction and impurity removal after adding the catalyst include:

[0059] Add the catalyst DMAP, heat to 45 - 55 °C and react overnight. Subsequently, evaporate the solvent, add dilute hydrochloric acid to precipitate the solid, and then filter, wash with water and dry in sequence.

[0060] By way of illustration and not limitation, in step S2, the crosslinking agent is EDC.HCl; the reaction and impurity removal after adding the crosslinking agent include:

[0061] Add EDC.HCl and react at room temperature for more than 3 h; subsequently, evaporate the solvent, perform extraction and drying, and then purify by column chromatography.

[0062] By way of illustration and not limitation, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

[0063] In one of the embodiments, the preparation method is specifically as follows:

[0064] Provide dehydroepiandrosterone and succinic anhydride, add them to DMF, add the catalyst DMAP, heat and react at 50 °C overnight, evaporate the solvent, add dilute hydrochloric acid to precipitate the solid, filter, wash with water and dry to obtain dehydroepiandrosterone carboxylic acid derivative.

[0065] Add the dehydroepiandrosterone carboxylic acid derivative, HOSu and EDC.HCl to DMF, react at room temperature for more than 3 h, evaporate the solvent, add an appropriate amount of water and dichloromethane for extraction, dry the organic phase with anhydrous sodium sulfate, and purify by column chromatography to obtain dehydroepiandrosterone carboxylic acid NHS active ester.

[0066] The applicant found that the above-mentioned dehydroepiandrosterone carboxylic acid NHS active ester is an active intermediate and can be used as a marker for binding to proteins or other molecules. Specifically, the NHS active ester is a chemical group that can form a stable amide bond with a compound containing a primary amine (-NH2). Therefore, the dehydroepiandrosterone carboxylic acid NHS active ester of the present application can be used to create markers with specific functions, which can then be used in bioanalysis applications such as chemiluminescence immunoassay. Markers generally refer to those substances that can be recognized or measured during an experiment or detection, such as fluorescent dyes, enzymes or radioactive isotopes, etc.

[0067] For example, the dehydroepiandrosterone carboxylic acid NHS active ester is configured to act as a marker precursor, providing an active site to achieve effective binding with BSA, thereby synthesizing the DHEA - BSA - AE marker.

[0068] Specifically, it includes:

[0069] Provide the above-mentioned dehydroepiandrosterone carboxylic acid NHS active ester marker;

[0070] The dehydroepiandrosterone carboxylic acid NHS active ester label and AE(NSP-SA-NHS) are used to label BSA to obtain the DHEA-BSA-AE label.

[0071] By way of illustration and not limitation, the use of the dehydroepiandrosterone carboxylic acid NHS active ester label and AE(NSP-SA-NHS) to label BSA includes:

[0072] The DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester is added to the PB solution of BSA, and after reaction, it is purified to obtain the DHEA-BSA label;

[0073] The DMF solution of the AE(NSP-SA-NHS) is added to the PB solution of the DHEA-BSA label, and after reaction, it is purified to obtain the DHEA-BSA-AE label.

[0074] By way of illustration and not limitation, before adding the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester to the PB solution of BSA, it further includes:

[0075] Dissolve the dehydroepiandrosterone carboxylic acid NHS active ester in DMF to prepare a 2 mg / ml solution;

[0076] Dissolve AE(NSP-SA-NHS) in DMF to prepare a 4 mg / ml solution;

[0077] Dissolve BSA in 0.1M PB pH7.4 to prepare a 1 mg / ml solution.

[0078] Specifically, in one embodiment, the addition of the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester to the PB solution of BSA, and after reaction, purification to obtain the DHEA-BSA label includes:

[0079] According to the labeling ratio of the dehydroepiandrosterone carboxylic acid NHS active ester to BSA, an appropriate amount of the DMF solution of the dehydroepiandrosterone carboxylic acid NHS active ester is added to the PB solution of BSA, shaken at room temperature for 1.5 h, and then dialyzed and purified 3 times with 0.1M PB pH 7.4 to obtain the DHEA-BSA label.

[0080] Specifically, in one embodiment, the addition of the DMF solution of the AE(NSP-SA-NHS) to the PB solution of the DHEA-BSA label, and after reaction, purification to obtain the DHEA-BSA-AE label includes:

[0081] According to the labeling ratio of AE (NSP-SA-NHS) and DHEA-BSA, an appropriate amount of the DMF solution of AE was taken and added to the PB buffer solution of DHEA-BSA, and the mixture was shaken and reacted for 1.5 h. Then, it was dialyzed and purified three times with 0.1 M PB pH 6.8 to obtain the DHEA-BSA-AE label, which is the key competitive antigen raw material for the chemiluminescent immunoassay reagent of dehydroepiandrosterone magnetic particles.

[0082] The present application will be further described in detail below in conjunction with specific embodiments.

[0083] This embodiment discloses a preparation method of the key competitive antigen raw material (DHEA-BSA-AE label) for the chemiluminescent immunoassay reagent of dehydroepiandrosterone magnetic particles, which includes the following steps:

[0084] S1. Provide dehydroepiandrosterone (1 g, 1 eq), succinic anhydride (1.4 g, 4 eq), add them to DMF (20 ml), add the catalyst DMAP (0.43 g, 1 eq), heat and react overnight at 50 °C, evaporate the solvent, add dilute hydrochloric acid to precipitate a solid, filter, wash with water, and dry to obtain the dehydroepiandrosterone carboxylic acid derivative, with a yield of 85%.

[0085] The mass spectrum of the obtained product is as Figure 1 shown. It can be seen from the figure that its mass-to-charge ratio (m / z) values are 386.95 (M-1) and 775.70 (2M-1).

[0086] S2. Add the dehydroepiandrosterone carboxylic acid derivative (0.5 g, 1 eq), Hosu (0.23 g, 1.5 eq), and EDC.HCl (0.37 g, 1.5 eq) to DMF (15 ml), react at room temperature for more than 3 h, evaporate the solvent, add an appropriate amount of water and dichloromethane for extraction, dry the organic phase with anhydrous sodium sulfate, and purify by column chromatography to obtain the dehydroepiandrosterone carboxylic acid NHS active ester, with a yield of 75%.

[0087] The mass spectrum of the obtained product is as Figure 2 shown. It can be seen from the figure that its mass-to-charge ratio (m / z) value is 484.10 (M-1). Since the carboxylic acid active ester bond energy is weak and is broken during mass spectrometry ionization, there are more carboxylic acid fragments 386.95 (M-1) removing NHS.

[0088] S3. Label BSA with dehydroepiandrosterone carboxylic acid NHS active ester and AE (NSP-SA-NHS):

[0089] Dissolve dehydroepiandrosterone carboxylic acid NHS active ester in DMF to prepare a 2 mg / ml solution; dissolve AE (NSP-SA-NHS) in DMSO to prepare a 4 mg / ml solution; dissolve BSA in 0.1 M PB pH 7.4 to prepare a 1 mg / ml solution.

[0090] According to the labeling ratio of dehydroepiandrosterone carboxylic acid NHS active ester to BSA, take an appropriate amount of the dehydroepiandrosterone carboxylic acid NHS active ester DMF solution and add it to the BSA PB solution, shake and react at room temperature for 1.5 h, and then dialyze and purify 3 times with 0.1 M PB pH 7.4 to obtain the DHEA-BSA conjugate.

[0091] According to the labeling ratio of AE (NSP-SA-NHS) and DHEA-BSA, take an appropriate amount of the AE DMF solution, add it to the DHEA-BSA PB buffer solution, shake and react for 1.5 h, and then dialyze and purify 3 times with 0.1 M PB pH 6.8 to obtain the DHEA-BSA-AE conjugate, which is the key competitive antigen raw material for the chemiluminescent immunoassay reagent of dehydroepiandrosterone sulfate magnetic particles.

[0092] Preparation method of biotin-labeled DHEA-S antibody:

[0093] Dissolve biotin-NHS active ester in DMSO to prepare a 4 mg / ml concentration; dissolve DHEA-S antibody in 0.1 M PB pH7.4 buffer solution to prepare a 1 mg / ml solution. According to the labeling ratio of biotin-NHS active ester to DHEA-S antibody, take an appropriate amount of the biotin-NHS active ester DMSO solution and add it to the DHEA-S antibody PB solution, shake and react at room temperature for 1.5 h, and then dialyze and purify 3 times with 0.1M PB pH 7.4 to obtain the biotin-labeled DHEA-S antibody.

[0094] In this example, the raw materials of the relevant kit are as follows:

[0095] Working calibrator target values: (0; 4.5; 15; 45; 450; 980) μg / dL.

[0096] Enterprise reference product target values: (72.6; 634.27) μg / dL.

[0097] Quality control product target values: (72.6; 634.27) μg / dL.

[0098] Streptavidin magnetic beads: Dilute streptavidin magnetic beads with magnetic bead buffer.

[0099] Reagent R1: Dilute the biotin-labeled DHEA-S antibody with reagent buffer.

[0100] Reagent R2: Dilute the DHEA-BSA-AE label with reagent buffer solution.

[0101] In this embodiment, the performance test was carried out with a chemiluminescence analyzer as follows:

[0102] 1) Appearance (reagent, calibrator, quality control product): Each component of the liquid is clear and transparent; the calibrator is a clear liquid without turbidity or precipitation; the quality control product should be a clear liquid without turbidity or precipitation.

[0103] 2) Accuracy: When detecting the enterprise reference product with the reagent kit, the relative deviation of the measurement result should not exceed ±10%.

[0104] Experimental method: Detect the enterprise reference products at the levels of (70±14) μg / dL and (625±125.0) μg / dL with the reagent kit, measure them 3 times repeatedly, record the average value of the results as M, and calculate the relative deviation B according to the formula:

[0105] Bi=(Xi-T) / T×100%

[0106] In the formula: Bi—relative deviation; Xi—average measured concentration; T—calibrated concentration.

[0107] The test results at 2-8°C refrigeration are shown in the following table:

[0108] Accuracy L1 L2 1 74.663 696.522 2 73.355 689.319 3 74.409 696.144 Mean 74.14 694.00 Target value 72.6 634.27 Relative deviation 2.12％ 9.42％

[0109] The test results of accelerating at 37°C for 7 days are as follows:

[0110] Accuracy L1 L2 1 68.866 632.364 2 68.396 650.991 3 69.106 620.190 Mean 68.79 634.52 Target value 72.6 634.27 Relative deviation -5.25％ 0.04％

[0111] 3) Minimum detectable limit

[0112] Experimental method: Use the zero-concentration calibrator or sample diluent as the sample for detection, measure it 20 times repeatedly, obtain the RLU values (relative luminescence values) of the 20 measurement results, calculate their average value (M) and standard deviation (SD), obtain M-2SD, and perform two-point regression fitting on the concentration-chemiluminescence (RLU) value results between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the RLU value corresponding to M-2SD into the equation to find the corresponding concentration value, which is the minimum detectable limit, as shown in the following table specifically.

[0113]

[0114]

[0115] 4) Linearity: In the range of [2.5-1000.0] μg / dL, the linear correlation coefficient (r) should not be lower than 0.9900.

[0116] Experimental method: Dilute high-value samples close to the upper limit of the linear range to at least 5 concentrations in a certain proportion, test each concentration point 3 times, establish the corresponding dose-response curve, and calculate the correlation coefficient (r).

[0117] Lot# (refrigerated at 2 - 8°C) Experimental data are shown in the following table:

[0118]

[0119]

[0120] Lot# (accelerated at 37°C for 7 days) Experimental data are shown in the following table:

[0121]

[0122] 5) Precision

[0123] Intra-batch precision: Samples at the concentration levels of (70.0 ± 14.0) μg / dL and (625 ± 125.0) μg / dL are detected, and the coefficient of variation (CV) of the measurement results should not be greater than 10%.

[0124] Experimental method: Use the same batch of kits to repeat the detection of samples at the concentrations of (70.0 ± 14.0) μg / dL and (625 ± 125.0) μg / dL 10 times each, calculate the average value (M) and standard deviation (SD) of the measurement results at each level. Calculate the coefficient of variation (CV) according to the formula:

[0125] CV = SD / M × 100%

[0126] Where: CV - coefficient of variation; SD - standard deviation; M - average value of the measurement results.

[0127] Lot# (refrigerated at 2 - 8°C) Experimental data are shown in the following table:

[0128] Intra-batch precision QC1 QC2 1 79.636 750.837 2 80.284 780.31 3 82.767 769.958 4 86.396 747.332 5 79.194 763.48 6 80.636 582.319 7 77.423 757.211 8 79.191 759.432 9 90.814 768.101 10 78.791 769.028 Mean 81.51 744.80 SD 4.118 57.912 CV 5.05％ 7.78％

[0129] Lot# (accelerated at 37°C for 7 days) Experimental data are shown in the following table:

[0130]

[0131]

[0132] Inter-batch precision: Use kits from 3 batches to detect samples at the concentration levels of (70.0 ± 14.0) μg / dL and (625 ± 125.0) μg / dL respectively, and the coefficient of variation (CV) of the measurement results should not be greater than 15%.

[0133] Experimental method: Samples with concentrations of (70.0 ± 14.0) μg / dL and (625 ± 125.0) μg / dL were detected using three kits with different batch numbers, each repeated 10 times. The mean (M) and standard deviation (SD) of the 30 measurement results for each concentration sample were calculated. The coefficient of variation (CV) was calculated according to the formula. The experimental data are shown in the following table:

[0134]

[0135]

[0136]

[0137] 6) Anti-interference: Hemoglobin (≤200 mg / dL), triglyceride (≤1500 mg / dL), bilirubin (≤30 mg / dL), biotin (≤30 ng / mL); the interference rate of substances within the above concentration ranges on the test results is ≤ ±10%.

[0138] Experimental method: Interferents were added to serum samples (or antigens) with concentration values but without interfering substances according to the corresponding ratios, and detected 3 times respectively. The mean value was calculated, and the result was compared with the result of the sample without interferents. If it is lower or higher than ±10% of the sample without interferents, it is considered that the sample is affected by the interferent, and the concentration of this interferent is the highest content at which the sample is affected by the interferent.

[0139] Lot# (refrigerated at 2 - 8°C) Experimental data are shown in the following table:

[0140]

[0141]

[0142]

[0143]

[0144] Lot# (accelerated at 37°C for 7 days) Experimental data are shown in the following table:

[0145]

[0146]

[0147]

[0148] 7) Sample correlation: 40 fresh serum samples covering the linear range were detected simultaneously with the control reagent, and the correlation coefficient R ≥ 0.975 was required.

[0149] Experimental method: Fresh serum samples covering the entire linear range were detected on the Roche fully automatic chemiluminescence analysis system and the Yingkai Shine i2910 instrument respectively, and the correlation coefficient R of the detection results of the two instruments was calculated. The relevant experimental data are shown in the following table and Figure 3 as follows:

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] As can be seen from the above table and Figure 3 it can be known that in this embodiment, when detecting dehydroepiandrosterone sulfate in the sample with the dehydroepiandrosterone sulfate antibody reagent, by comparing with the Roche sample, the correlation R 2 reaches 0.98.

[0157] To sum up: In this application, dehydroepiandrosterone is selected as the starting material for the competitive antigen. It has a wide source and low cost. Through two-step reactions, dehydroepiandrosterone carboxylic acid NHS active ester can be prepared, which can be used as an active intermediate and a marker for binding to proteins or other molecules.

[0158] Subsequently, it is coupled with the carrier protein BSA. The dehydroepiandrosterone derivative can be covalently linked to BSA, and after purification, it is labeled with acridinesulfonamide. After purification, the key competitive antigen raw material for the dehydroepiandrosterone magnetic particle chemiluminescence immunoassay reagent is obtained.

[0159] Moreover, since the chemical structures of dehydroepiandrosterone and dehydroepiandrosterone sulfate are extremely similar but not exactly the same, when it is used to bind to the dehydroepiandrosterone sulfate antibody, the affinity is neither greater than that of dehydroepiandrosterone sulfate in the sample nor too small, reaching an ideal affinity level. When detecting dehydroepiandrosterone sulfate in the sample with the dehydroepiandrosterone sulfate antibody reagent, by comparing with the Roche sample, the correlation R 2 reaches 0.98.

[0160] All the technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0161] The embodiments described above merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A dehydroepiandrosterone carboxylic acid NHS active ester marker, characterized in that: The dehydroepiandrosterone carboxylic acid NHS active ester marker has a structural formula shown in Formula 1: (Formula 1) 2. A method for preparing a dehydroepiandrosterone carboxylic acid NHS active ester marker, characterized in that: The preparation method comprises the following steps: S1, providing dehydroepiandrosterone and succinic anhydride, adding the dehydroepiandrosterone and succinic anhydride to an organic solvent, adding a catalyst, heating for reaction, and removing impurities to obtain a dehydroepiandrosterone carboxylic acid derivative; S2, adding the dehydroepiandrosterone carboxylic acid derivative and HOSu into an organic solvent, adding a cross-linking agent, reacting and removing impurities to obtain the dehydroepiandrosterone carboxylic acid NHS active ester.

3. The preparation method according to claim 2, characterized in that: In step S1, the catalyst is DMAP; and / or, The step of heating the reaction after adding the catalyst and removing impurities comprises: Add DMAP as a catalyst, heat to 45-55°C and react overnight, then evaporate the solvent, add dilute hydrochloric acid to precipitate the solid, and then filter, wash with water and dry it in sequence.

4. The preparation method according to claim 2, characterized in that: In step S2, the cross-linking agent is EDC.HCl; and / or, The reaction and impurity removal after adding the cross-linking catalyst comprises: Add EDC.HCl and react at room temperature for more than 3 hours; then evaporate the solvent, extract and dry, and purify by column.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

6. The dehydroepiandrosterone carboxylic acid NHS active ester marker obtained by the preparation method according to any one of claims 2 to 5.

7. Use of the dehydroepiandrosterone carboxylic acid NHS active ester marker according to claim 1 or 6 in the preparation of a dehydroepiandrosterone sulfate magnetic particle chemiluminescent immunoassay reagent or a dehydroepiandrosterone sulfate magnetic particle chemiluminescent immunoassay kit.

8. A method for preparing a chemiluminescent immunoassay reagent, characterized in that: include: Providing the dehydroepiandrosterone carboxylic acid NHS active ester marker according to claim 6; BSA was labeled with the dehydroepiandrosterone carboxylic acid NHS active ester label and AE (NSP-SA-NHS) to obtain DHEA-BSA-AE label.

9. The method for preparing the chemiluminescent immunoassay reagent according to claim 8, characterized in that: The method of labeling BSA with the dehydroepiandrosterone carboxylic acid NHS active ester label and AE (NSP-SA-NHS) comprises: Adding the dehydroepiandrosterone carboxylic acid NHS active ester DMF solution to the BSA PB solution, and purifying after reaction to obtain a DHEA-BSA marker; The DMF solution of AE (NSP-SA-NHS) is added to the PB solution of DHEA-BSA marker, and the DHEA-BSA-AE marker is obtained by purification after reaction.

10. The DHEA-BSA-AE marker obtained by the preparation method of the chemiluminescent immunoassay reagent according to claim 8 or 9.

Images