High-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit and preparation method thereof

Abstract

The invention relates to the technical field of biomedical detection, in particular to a high-sensitivity dehydroepiandrosterone sulfate electrochemical luminescence immunoassay detection kit and a preparation method thereof. The invention relates to a high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit. The kit comprises a streptavidin magnetic bead reagent, a biotin-labeled dehydroepiandrosterone sulfate antibody reagent, a terpyridyl ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent and excitation liquid, the high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit prepared by the invention has a good linear range, accuracy, sensitivity, specificity, precision and stability.

Description

Technical Field

[0001] This application relates to the field of biomedical detection technology, and in particular to a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate and its preparation method. Background Technology

[0002] Dehydroepiandrosterone sulfate (DHEA-S) is the most abundant steroid hormone in the human bloodstream, primarily secreted by the zona reticularis of the adrenal cortex. Detection of its levels is of crucial clinical value for assessing adrenal cortical function, diagnosing polycystic ovary syndrome, congenital adrenal hyperplasia, Cushing's syndrome, and certain endocrine-related tumors.

[0003] In the field of biomedical testing, immunoassay techniques targeting the biomarker DHEA-S have been continuously iterated to meet evolving needs, with the development of techniques such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay. However, existing technologies still have significant shortcomings in terms of sensitivity, stability, detection efficiency, and industrial adaptability, making it difficult to meet the high standards required for clinical diagnosis and market applications. The specific current status and limitations of these technologies are as follows: 1. Radioimmunoassay As a classic early immunoassay technique, it relies on radioactive markers to achieve quantitative analysis of antigen-antibody reactions. However, this method has fundamental application defects: the detection process is accompanied by radioactive contamination, which places extremely high demands on the operating environment, protective equipment, and personnel qualifications; the reagents have a very short shelf life due to radioactive decay, requiring frequent replacement to ensure the reliability of the test; and the operation process is cumbersome and complex, making it impossible to achieve large-scale and standardized testing through automated equipment, resulting in low overall efficiency.

[0004] 2. Enzyme-linked immunosorbent assay (ELISA) This technology uses enzyme-catalyzed substrate color development to achieve quantitative detection, which avoids the risk of radioactive contamination. However, its detection performance and practicality still have significant shortcomings: low sensitivity, narrow linear detection range, and insufficient detection accuracy for low-concentration samples; redundant operation steps and long individual detection cycles; more importantly, the stability of detection results is easily affected by factors such as ambient temperature and reaction time deviation, making it difficult to guarantee intra-batch and inter-batch precision.

[0005] 3. Existing chemiluminescence method With its high sensitivity and automated detection capabilities, chemiluminescence has become the mainstream technology in the current immunoassay market; however, for the detection of specific biomarkers such as DHEA-S, existing chemiluminescence products still have room for improvement in many dimensions, such as: Sensitivity and specificity: The high cross-reactivity rate of the detection antibody with structural analogues can easily lead to non-specific binding, resulting in inaccurate detection of low-concentration samples or weak ability to identify interfering substances. Reaction stability: The luminescence signal decays rapidly, the effective detection window is short, making it difficult to guarantee the consistency of detection results between different batches and within the same batch, thus limiting precision; Reagent stability: The core components of the reagent kit, such as enzyme labels and luminescent substrates, are prone to activity degradation during long-term storage or transportation, which directly affects the shelf life of the reagents and the reliability of the detection. Detection speed: The kinetics of antigen-antibody reactions are slow, and the total detection time is difficult to match the needs of rapid clinical diagnosis.

[0006] Furthermore, the existing DHEA-S chemiluminescence detection reagent also uses an acridinium ester chemiluminescence platform. Since acridinium ester is a flash-type luminescence system, the capture of the luminescence signal is highly dependent on precision instruments, resulting in insufficient precision, which makes the reagent kit unable to meet market demands.

[0007] Therefore, how to effectively solve the problems of insufficient sensitivity and specificity, poor stability, and slow detection speed of existing technologies in the field of DHEA-S detection has become an urgent issue to be addressed. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this application provides a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate and its preparation method.

[0009] This application discloses a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate and its preparation method. The kit uses a signal amplification process of streptavidin and biotin, a high-efficiency ruthenium tripyridine labeling system, and optimized signal amplification technology. The limit of detection (LoD) of this kit can reach 0.05 µg / dL, which can accurately detect low levels of normal and pathologically reduced levels of dehydroepiandrosterone sulfate (DHEA-S), meeting the stringent requirements of pediatrics, endocrinology, and other departments for low-concentration detection.

[0010] This application discloses a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate and its preparation method. The kit has high sensitivity, a wide linear range, extremely low cross-reactivity, and long-lasting reagent stability.

[0011] In the first aspect, this application provides a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate, employing the following technical solution: A high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate (DHEA-S), the kit comprising: Streptavidin magnetic bead reagent; Biotin-labeled dehydroepiandrosterone sulfate antibody reagent; Tripyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent; and... Activation solution.

[0012] Preferably, the streptavidin magnetic bead reagent comprises streptavidin magnetic beads and a magnetic bead diluent; The magnetic bead diluent comprises the following raw materials: neutral buffer 1, methyl-β-cyclodextrin, stabilizer 1, surfactant 1, and preservative 1; Preferably, the neutral buffer 1 comprises at least one of HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 1 is 7-7.5; Preferably, the stabilizer 1 includes at least one of BSA, casein, and fish gelatin; Preferably, the surfactant 1 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 1 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the magnetic bead diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 1% (w / v)-3% (w / v) methyl-β-cyclodextrin, 0.1% (w / v)-1% (w / v) BSA, 0.1% (w / v)-1% (w / v) Tween-20, and 0.05% (w / v)-0.15% (w / v) Proclin 300.

[0013] By adopting the above technical solution, this application improves the formulation of the magnetic bead diluent by adding methyl-β-cyclodextrin to the diluent. The hydrophobic cavity of methyl-β-cyclodextrin can selectively encapsulate free hydrophobic steroid hormones (such as DHEA) in the sample without affecting the highly polar DHEA-S. This effectively eliminates the background noise caused by non-specific adsorption or interference of DHEA during sample processing, making it suitable for the accurate detection of low concentrations of DHEA-S and effectively improving reagent sensitivity.

[0014] Preferably, the ruthenium-labeled terpyridine sulfate dehydroepiandrosterone derivative reagent comprises a DHEA-S antigen derivative terpyridine ruthenium label and a ruthenium diluent; The ruthenium diluent comprises the following raw materials: neutral buffer 2, pyridoxine hydrochloride, stabilizer 2, sugar protectant, surfactant 2, and preservative 2; Preferably, the neutral buffer 2 includes one or more of HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 2 is 7-7.5; Preferably, the stabilizer 2 includes at least one of BSA, casein, and fish gelatin; Preferably, the sugar protectant includes at least one of sucrose, trehalose, and dextran; Preferably, the surfactant 2 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 2 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT.

[0015] Preferably, the ruthenium diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 0.1% (w / v)-0.5% (w / v) pyridoxine hydrochloride, 0.5% (w / v)-1.5% (w / v) BSA, 3% (w / v)-7% (w / v) trehalose, 0.05% (w / v)-0.3% (w / v) Tween-20, and 0.05% (w / v)-0.3% (w / v) Proclin 300.

[0016] By adopting the above technical solution, since chemiluminescent markers are easily oxidized and quenched during long-term storage, this application improves the ruthenium diluent raw material by introducing pyridoxine hydrochloride as a specific free radical scavenger. The added pyridoxine hydrochloride can not only effectively quench singlet oxygen, but also form a weak complex with the metal ions in the formulation, preventing the degradation of the markers caused by metal ion catalysis. Compared with the conventional formulation, it effectively improves the thermal stability of the reagent and extends the shelf life of the kit.

[0017] Preferably, the activation solution comprises the following raw materials: neutral buffer solution 3, pH adjustment aid, salt substance, surfactant 3, and preservative 3; Preferably, the neutral buffer 3 is a piperazine-N,N'-bis(ethanesulfonic acid) buffer, and the pH of the neutral buffer 3 is 7-7.5; Preferably, the pH adjusting agent is tripropylamine; Preferably, the salt substance includes at least one of sodium chloride, potassium chloride, and magnesium chloride; Preferably, the surfactant 3 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 3 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT.

[0018] Preferably, the activation solution comprises the following raw materials: 0.05 mol / L-0.2 mol / L piperazine-N,N'-bis(ethanesulfonic acid), 0.05 mol / L-0.2 mol / L tripropylamine, 0.1 mol / L-0.2 mol / L sodium chloride, 0.05% (v / v)-0.15% (v / v) Triton X-100, and 0.01% (w / v)-0.1% (w / v) Proclin 300.

[0019] By adopting the above technical solution, this application improves the raw materials of the excitation solution by using a piperazine-N,N'-bis(ethanesulfonic acid) (PIPES) buffer system, which has a stable zwitterionic structure and metal chelating ability, and contains a free radical scavenger and a metal ion chelator. The added PIPES buffer provides a more stable coordination environment for ruthenium tripyridine. The combination with other raw materials can efficiently quench free radicals generated by trace metal ions or photocatalysis during storage, effectively improving the stability of the excitation solution.

[0020] Preferably, the kit further includes calibrators, quality control products, and washing solution.

[0021] Preferably, the calibrator includes a dehydroepiandrosterone sulfate calibrator and a working diluent; the quality control includes a dehydroepiandrosterone sulfate quality control and a working diluent. The working diluent includes the following ingredients: neutral buffer 4, protein stabilizer, L-proline, trehalose, surfactant 4, and preservative 4. Preferably, the neutral buffer 4 includes one or more of MES buffer, HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 4 is 6-6.5. Preferably, the protein stabilizer includes at least one of glycerol, bovine serum, and polyethylene glycol; Preferably, the surfactant 4 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 4 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT.

[0022] The working diluent comprises the following ingredients: 40 mmol / L-60 mmol / L MES, 10% (v / v)-20% (v / v) bovine serum, 0.1% (v / v)-1% (v / v) L-proline, 1% (v / v)-10% (v / v) trehalose, 0.05% (v / v)-0.3% (v / v) Tween-20, 1% (v / v)-10% (v / v) glycerol, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

[0023] By adopting the above technical solution, this application optimizes the protein stabilization system by using a complex of L-proline and trehalose. L-proline, as an osmotic pressure compensating solute, can more effectively maintain the native conformation of the antibody in the liquid environment through preferential repulsion. In synergy with trehalose, it effectively improves the stability of calibrators and quality control products, meeting the needs of transportation and use in high-temperature regions.

[0024] Preferably, the washing liquid comprises the following raw materials: trisodium phosphate, phosphoric acid, salt substances, surfactant 5, and preservative 5; Preferably, the salt substance includes at least one of sodium chloride, potassium chloride, and magnesium chloride; Preferably, the surfactant 5 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 5 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT.

[0025] Preferably, the washing solution comprises the following raw materials: 1% (v / v)-2% (v / v) trisodium phosphate, 1% (v / v)-2% (v / v) phosphoric acid, 15% (v / v)-20% (v / v) sodium chloride, 1% (v / v)-2% (v / v) Tween-20, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

[0026] By adopting the above technical solution, this application discloses a buffer-stabilizer system, which effectively avoids the easy crystallization and precipitation of highly concentrated detergent at low temperatures, thereby achieving the best balance between cost and stability while maintaining ultra-high detergent performance.

[0027] Secondly, this application provides a method for preparing a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate, using the following technical solution: A method for preparing a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate includes the following preparation steps: providing streptavidin magnetic bead reagent, biotin-labeled dehydroepiandrosterone sulfate antibody reagent, ruthenium tripyridine-labeled dehydroepiandrosterone sulfate derivative reagent, calibrators, quality control materials, washing solution, and activation solution to prepare the assay kit.

[0028] Preferably, the biotin-labeled dehydroepiandrosterone sulfate antibody reagent comprises a biotin-labeled dehydroepiandrosterone sulfate antibody; The preparation method of the biotin-labeled dehydroepiandrosterone sulfate antibody includes the following steps: DHEA-S monoclonal antibody and biotin are coupled together, and then a blocking agent is added to carry out the reaction to obtain the biotin-labeled dehydroepiandrosterone sulfate antibody. Preferably, the molar ratio of the DHEA-S monoclonal antibody to biotin is 1:(5-20). Preferably, when conjugating DHEA-S monoclonal antibody and biotin, the reaction system is protected from light and incubated at room temperature for 60-90 minutes.

[0029] Preferably, the amount of the sealing agent added is 40-60 μL.

[0030] Preferably, a blocking agent is added for the reaction, and the reaction time is incubation at room temperature for 30-40 minutes.

[0031] Preferably, the ruthenium-labeled terpyridine sulfate dehydroepiandrosterone derivative reagent comprises a DHEA-S antigen derivative terpyridine ruthenium label and a ruthenium diluent; The preparation method of the DHEA-S antigen derivative terpyridine ruthenium label is as follows: the DHEA-S-BSA conjugate is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to obtain the activated DHEA-S-BSA conjugate with a final concentration of 5 mmol / L-20 mmol / L. According to the molar ratio of ruthenium to protein (10-30:1), NHS-Ru(bpy)3 2+ The solution was added to the activated DHEA-S-BSA conjugate to carry out the coupling reaction, and then a blocking agent was added to carry out the reaction to obtain the DHEA-S antigen derivative terpyridine ruthenium-labeled product. Preferably, the DHEA-S-BSA conjugate is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) at a temperature of 20-25°C and incubated for 20-30 minutes.

[0032] Preferably, NHS-Ru(bpy)3 2+The solution was added to the activated DHEA-S-BSA conjugate and incubated with gentle stirring or rotation at room temperature (20-25℃) for 120 min.

[0033] Preferably, the amount of the sealing agent added is 40-60 μL.

[0034] Preferably, a blocking agent is added to initiate the reaction, and the mixture is incubated at room temperature for 30-40 minutes.

[0035] By adopting the above technical solution, this application uses a covalent coupling technique after the antigen is activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0036] This application activates the carboxyl group of the derivative and reacts it with ruthenium terpyridine in an aqueous phase at room temperature and neutral pH. The reaction proceeds almost quantitatively with very few byproducts, is not affected by other functional groups, and achieves a labeling yield of over 90%, exhibiting high selectivity and high efficiency.

[0037] The reaction conditions described in this application are mild, avoiding harsh conditions such as strong acids, strong alkalis, or high temperatures, effectively protecting the stability of the DHEA-S antigen and the ruthenium terpyridine luminescent group and preventing their degradation during the labeling process.

[0038] This application covalently binds amide bonds, resulting in a well-defined and stable structure. The resulting labeled complex is a stoichiometric, homogeneous, and single-oriented product, which is fundamentally different from the mixtures obtained by conventional methods, laying a chemical foundation for batch-to-batch consistency of the kit.

[0039] The terpyridine ruthenium-labeled dehydroepiandrosterone sulfate derivative prepared in this application has excellent stability, chemically stable linkages and a sterically stable structure, which significantly prolongs the activity retention time of the labeled complex when stored at 4℃ or -20℃ and has a low luminescence signal attenuation rate.

[0040] Based on high immunoreactivity, this application combines the high sensitivity and low background advantages of ruthenium tripyridine electrochemiluminescence, enabling the DHEA-S immunoassay method established using the label of this application to have the following characteristics: Lower detection limit: It can detect lower concentrations of DHEA-S.

[0041] Wider linear range: less likely to exhibit "hook effect" in high concentration range.

[0042] Higher precision: significantly reduced intra-batch and inter-batch coefficients of variation.

[0043] Better specificity: lower cross-reactivity with other structurally similar steroid hormones (such as DHEA and androstenedione).

[0044] In summary, this application includes at least one of the following beneficial technical effects: This application discloses a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate and its preparation method. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate prepared in this application has good linear range, accuracy, sensitivity, specificity, precision and thermal stability. Attached Figure Description

[0045] Figure 1 A linear range curve graph; Figure 2 This is a correlation graph between clinical serum and Roche reagents. Detailed Implementation

[0046] The technical solutions of this application are further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0047] All raw materials involved in this application are commercially available products, among which, Sulfo-NHS-Biotin, CAS:119616-38-5; Methyl-β-cyclodextrin and pyridoxine hydrochloride were purchased from Merck. DHEA-S monoclonal antibody, manufacturer: Merck (Sigma-Aldrich), catalog number: MABN1007; DHEA-S-BSA coupling, manufacturer: Merck (Sigma-Aldrich), part number: A8610; NHS-Ru(bpy)3 2+ Solution: Manufacturer: Sigma-Aldrich (Merck); Ru(bpy)3 2+ The parent compound is CAS number 14323-06-9; NHS ester is its derivative. Bovine serum, purchased from Thermo Fisher Scientific; L-proline, purchased from Aladdin; Piperazine-N,N'-bis(ethanesulfonic acid), CAS:100037-69-2; The present application will be further described in detail below with reference to embodiments and comparative examples.

[0048] A high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate, the kit comprising: Streptavidin magnetic bead reagent; Biotin-labeled dehydroepiandrosterone sulfate antibody reagent; Tripyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent; DHEA-S calibrators; solutions containing different known concentrations of DHEA-S for plotting standard curves, with human serum or buffer as the matrix.

[0049] DHEA-S quality control products; solutions containing different known concentrations of DHEA-S, used to control batch-to-batch variability, with human serum or buffer as the matrix.

[0050] Concentrated detergent; and Activation solution.

[0051] A method for preparing a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate includes the following steps: (1) Preparation of streptavidin magnetic bead reagent: The magnetic beads are Dynabeads™ M-280 streptavidin magnetic beads (2.8μm particle size, 10mg / ml).

[0052] In one specific feasible implementation, the magnetic beads are diluted to 0.8 mg / ml with a magnetic bead diluent before use. This diluent formulation ensures the long-term stability of the magnetic bead reagent.

[0053] The magnetic bead diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 1% (w / v)-3% (w / v) methyl-β-cyclodextrin, 0.1% (w / v)-1% (w / v) BSA, 0.1% (w / v)-1% (w / v) Tween-20, and 0.05% (w / v)-0.15% (w / v) Proclin 300.

[0054] In one specific implementation, the magnetic bead diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 2% (w / v) methyl-β-cyclodextrin, 0.5% (w / v) BSA, 0.5% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0055] (2) Preparation of biotin-labeled dehydroepiandrosterone sulfate antibody reagent The biotin-labeled dehydroepiandrosterone sulfate antibody reagent includes a biotin-labeled dehydroepiandrosterone sulfate antibody and a biotin diluent. The preparation method of biotin-labeled dehydroepiandrosterone sulfate antibody is as follows: DHEA-S monoclonal antibody and biotin are coupled together, and then a blocking agent is added to carry out the reaction to obtain biotin-labeled dehydroepiandrosterone sulfate antibody. Specifically, take the purified DHEA-S monoclonal antibody (concentration greater than 1-5 mg / mL, dissolved in 0.01M PBS, pH 7.4), and use ultrafiltration centrifugation or dialysis to replace the antibody in 0.1M phosphate buffer (pH 8) at 2-8℃, finally adjusting the antibody concentration to 1-2 mg / mL to obtain the antibody solution.

[0056] According to the molar ratio of DHEA-S monoclonal antibody to biotin of 1:(5-20), weigh biotin (Sulfo-NHS-Biotin), dissolve Sulfo-NHS-Biotin in an appropriate amount of ultrapure water, and prepare the solution immediately before use to obtain a biotin reagent solution.

[0057] At room temperature (20-25℃), slowly add the freshly prepared biotin reagent solution to the antibody solution while gently vortexing or stirring to mix. Protect the reaction system from light and incubate at room temperature (20-25℃) for 60-90 minutes.

[0058] After the reaction is complete, add 40-60 μL of a 1M glycine solution (pH 8.0) and incubate at room temperature for 30 minutes to quench any unreacted active ester groups.

[0059] Purification-dialysis method: Transfer the reaction solution to a pretreated dialysis bag and dialyze against 2L of storage buffer at 2-8℃. Change the buffer every 2 hours for a total of 4 dialysis cycles.

[0060] After dialysis, the above reaction solution is taken out, stabilizer and antifreeze are added, and after thorough mixing, it is dispensed in small doses and stored at -20℃ for a long time.

[0061] The biotin diluent consists of the following ingredients: 5 mmol / L-15 mmol / L PBS (pH 7.4), 0.5% (w / v)-1.5% (w / v) BSA, 1% (w / v)-10% (w / v) trehalose, 0.1% (w / v)-0.5% (w / v) Tween-20, and 0.05% (w / v)-0.2% (w / v) Proclin 300.

[0062] In one specific feasible implementation, the biotin diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 1% (w / v) BSA, 5% (w / v) trehalose, 0.2% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0063] (3) Preparation of terpyridine ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent The ruthenium-labeled terpyridine sulfate dehydroepiandrosterone derivative reagent includes the DHEA-S antigen derivative terpyridine ruthenium label and ruthenium diluent; The preparation method of the DHEA-S antigen derivative terpyridine ruthenium label is as follows: the DHEA-S-BSA conjugate is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide to obtain the activated DHEA-S-BSA conjugate with a final concentration of 5 mmol / L-20 mmol / L. According to the molar ratio of ruthenium to protein (10-30:1), NHS-Ru(bpy)3 2+ The solution was added to the activated DHEA-S-BSA conjugate to carry out the coupling reaction, and then a blocking agent was added to carry out the reaction to obtain the DHEA-S antigen derivative terpyridine ruthenium-labeled product. Specifically, the preparation method of the DHEA-S antigen derivative terpyridine ruthenium-labeled product is as follows: Dissolve the DHEA-S-BSA conjugate in PBS, and use an ultrafiltration centrifuge tube or dialysis method to replace the solution in 100 mmol / L PBS buffer (pH 8.0) at 2-8℃, finally adjusting the protein concentration to 2 mg / mL; this step is crucial as it removes free amino groups.

[0064] Add freshly prepared EDC solution to the protein solution to achieve a final concentration of 5 mmol / L-20 mmol / L. Mix gently and incubate at room temperature (20-25℃) for 20-30 minutes to activate the carboxyl groups on the protein.

[0065] According to the molar ratio of ruthenium to protein (10-30):1, NHS-Ru(bpy)3 2+ The solution is added to the activated protein solution.

[0066] Place the reaction mixture under light-protected conditions and incubate at room temperature (20-25℃) with gentle stirring or rotation for 120 min; add 40-60 μL of pre-cooled 1M glycine solution (pH 8.0) and continue incubation at room temperature for 30 min to quench unreacted EDC and active esters.

[0067] Purification-dialysis method: Transfer the reaction solution to a pretreated dialysis bag and dialyze against 2L of storage buffer at 2-8℃. Change the buffer every 2 hours for a total of 4 dialysis cycles until no free ruthenium signal can be detected in the dialysate (which can be detected by UV absorption or ECL background).

[0068] After dialysis, remove the above reaction solution, add a stabilizer, mix thoroughly, and then aliquot in small quantities and store at 2-8℃ for a long period of time, protected from light.

[0069] Preferably, the ruthenium diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 0.1% (w / v)-0.5% (w / v) pyridoxine hydrochloride, 0.5% (w / v)-1.5% (w / v) BSA, 3% (w / v)-7% (w / v) trehalose, 0.05% (w / v)-0.3% (w / v) Tween-20, and 0.05% (w / v)-0.3% (w / v) Proclin 300.

[0070] In one specific implementation, the ruthenium diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 0.2% (w / v) pyridoxine hydrochloride, 1% (w / v) BSA, 5% (w / v) trehalose, 0.2% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0071] (4) Preparation of DHEA-S calibrators The calibrators include dehydroepiandrosterone sulfate calibrator and working diluent; In one specific feasible implementation, when used, high-purity DHEA-S is diluted with working diluent to prepare a calibrator set containing 8 concentration points (0.1, 0.2, 4, 20, 100, 500, 1000, 1200 µg / dL).

[0072] The working diluent comprises the following ingredients: 40 mmol / L-60 mmol / L MES, 10% (v / v)-20% (v / v) bovine serum, 0.1% (v / v)-1% (v / v) L-proline, 1% (v / v)-10% (v / v) trehalose, 0.05% (v / v)-0.3% (v / v) Tween-20, 1% (v / v)-10% (v / v) glycerol, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

[0073] In one specific implementation, the working diluent comprises the following ingredients: 50 mmol / L MES (pH 6.0), 15% (v / v) bovine serum, 0.5% (v / v) L-proline, 5% (v / v) trehalose, 0.15% (v / v) Tween-20, 5% (v / v) glycerol, and 0.2% (v / v) Proclin 300.

[0074] (5) Preparation of DHEA-S quality control materials The quality control materials include dehydroepiandrosterone sulfate quality control material and working diluent; In one specific feasible implementation, when used, high-purity DHEA-S is diluted with a working diluent to prepare quality control samples containing 5 µg / dL and 500 µg / dL.

[0075] The working diluent comprises the following ingredients: 40 mmol / L-60 mmol / L MES, 10% (v / v)-20% (v / v) bovine serum, 0.1% (v / v)-1% (v / v) L-proline, 1% (v / v)-10% (v / v) trehalose, 0.05% (v / v)-0.3% (v / v) Tween-20, 1% (v / v)-10% (v / v) glycerol, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

[0076] In one specific implementation, the working diluent comprises the following ingredients: 50 mmol / L MES (pH 6.0), 15% (v / v) bovine serum, 0.5% (v / v) L-proline, 5% (v / v) trehalose, 0.15% (v / v) Tween-20, 5% (v / v) glycerol, and 0.2% (v / v) Proclin 300.

[0077] (6) Concentrated detergent; The washing solution comprises the following raw materials: 1% (v / v)-2% (v / v) trisodium phosphate, 1% (v / v)-2% (v / v) phosphoric acid, 15% (v / v)-20% (v / v) sodium chloride, 1% (v / v)-2% (v / v) Tween-20, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

[0078] In one specific implementation, the washing liquid comprises the following ingredients: 1.21% (v / v) trisodium phosphate, 1.53% (v / v) phosphoric acid, 17.25% (v / v) sodium chloride, 1.5% (v / v) Tween-20, and 0.2% (v / v) Proclin 300.

[0079] (7) Preparation of activating solution The activation solution comprises the following raw materials: 0.05 mol / L-0.2 mol / L piperazine-N,N'-bis(ethanesulfonic acid), 0.05 mol / L-0.2 mol / L tripropylamine, 0.1 mol / L-0.2 mol / L sodium chloride, 0.05% (v / v)-0.15% (v / v) Triton X-100, and 0.01% (w / v)-0.1% (w / v) Proclin 300.

[0080] In one specific embodiment, the activation solution comprises the following ingredients: 0.1 mol / L piperazine-N,N'-bis(ethanesulfonic acid) (PIPES buffer), 0.1 mol / L tripropylamine, 0.15 mol / L sodium chloride, 0.1% (v / v) Triton X-100, and 0.05% (w / v) Proclin 300.

[0081] Example 1:

[0082] A method for preparing a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate includes the following steps: 1. Preparation of streptavidin magnetic beads: The magnetic beads are Dynabeads™ M-280 streptavidin magnetic beads (2.8μm particle size, 10mg / ml).

[0083] 2. Preparation of biotin-labeled dehydroepiandrosterone sulfate antibody Take 1 mg of purified DHEA-S monoclonal antibody (concentration greater than 1-5 mg / mL, dissolved in 0.01 M PBS, pH 7.4), and ultrafilter the antibody storage buffer through a 30 kDa ultrafiltration tube until it is replaced with 0.1 M phosphate buffer (pH 8). Ultrafiltration conditions: 10000 rpm, 8 min, ultrafiltration twice consecutively, temperature 2-8℃. Finally, adjust the antibody concentration to 2 mg / mL to obtain the antibody solution.

[0084] According to the molar ratio of DHEA-S monoclonal antibody to biotin of 1:10, weigh 60 μg of Sulfo-NHS-Biotin, dissolve it in 1 mL of ultrapure water, and prepare the biotin reagent solution immediately before use.

[0085] At room temperature (20-25℃), slowly add freshly prepared biotin reagent solution to the antibody solution while stirring and mixing. Protect the reaction system from light and incubate at room temperature (20-25℃) for 60 minutes.

[0086] After the reaction was completed, 50 μL of a 1 M glycine solution (pH 8.0) was added and incubated at room temperature (20-25℃) for 30 minutes to quench any unreacted active ester groups.

[0087] Transfer the reaction solution to a pretreated dialysis bag and dialyze against 2L of storage buffer at 2-8℃. Change the buffer every 2 hours for a total of 4 dialysis cycles.

[0088] After dialysis, remove the above reaction solution, add 0.1% (w / v) BSA and 50% (w / v) glycerol, mix thoroughly, dispense in small doses, and store at -20℃ for long-term storage.

[0089] 3. Preparation of DHEA-S antigen derivatives labeled with terpyridine and ruthenium The preparation method of DHEA-S antigen derivative terpyridine ruthenium-labeled compound is as follows: Take 1.0 mg of DHEA-S-BSA conjugate (dissolved in PBS buffer), and ultrafilter the antibody storage buffer through a 30 kDa ultrafiltration tube until it is replaced with 100 mM PBS buffer (pH 8.0). Ultrafiltration conditions: 10000 rpm, 8 min, repeat twice, temperature 2-8℃. Finally, adjust the protein concentration to 2 mg / mL and remove free amino groups.

[0090] Add 50 μL of freshly prepared EDC solution (concentration 10 mg / ml) to the above protein solution to bring the final protein concentration to 15 mmol / L. Mix gently and incubate at room temperature (20-25℃) for 25 minutes to activate the carboxyl groups on the protein to obtain the activated protein solution.

[0091] According to the molar ratio of ruthenium to protein of 20:1, take NHS-Ru(bpy)3 containing 5 mmol of ruthenium(II). 2+ Add 100 μL of the solution to the activated protein solution.

[0092] Place the reaction mixture under light-protected conditions and gently stir at room temperature (20-25℃) for 120 min; add 50 μL of pre-cooled 1M glycine solution (pH 8.0) and continue incubation at room temperature for 30 min to quench unreacted EDC and active esters.

[0093] Purification-dialysis method: Transfer the reaction solution to a pretreated dialysis bag and dialyze against 2L of storage buffer at 2-8℃. Change the buffer every 2 hours for a total of 4 dialysis cycles until no free ruthenium signal can be detected in the dialysate (which can be detected by UV absorption or ECL background).

[0094] After dialysis, remove the above reaction solution, add 0.1% (w / v) BSA to the solution, mix thoroughly, aliquot in small quantities, and store at 2-8℃ for a long time, protected from light.

[0095] Kit assembly The streptavidin magnetic beads, biotin-labeled dehydroepiandrosterone sulfate antibody, and DHEA-S antigen derivative triple pyridine ruthenium-labeled reagent prepared above were assembled into a kit as follows: 4.1 Streptavidin magnetic bead reagent; Streptavidin magnetic bead reagent includes streptavidin magnetic beads and magnetic bead diluent; The magnetic bead diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 2% (w / v) methyl-β-cyclodextrin, 0.5% (w / v) BSA, 0.5% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0096] When using magnetic beads, dilute them to 0.8 mg / ml with magnetic bead diluent.

[0097] 4.2 Biotin-labeled dehydroepiandrosterone sulfate antibody reagent; The biotin-labeled dehydroepiandrosterone sulfate antibody reagent includes a biotin-labeled dehydroepiandrosterone sulfate antibody and a biotin diluent. The biotin diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 1% (w / v) BSA, 5% (w / v) trehalose, 0.2% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0098] When using this product, the biotin-labeled dehydroepiandrosterone sulfate antibody should be diluted to 0.8 μg / mL using biotin dilution buffer.

[0099] 4.3. Tripyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent The ruthenium-labeled terpyridine sulfate dehydroepiandrosterone derivative reagent includes a DHEA-S antigen derivative terpyridine ruthenium label and a ruthenium diluent; The ruthenium diluent comprises the following ingredients: 10 mmol / L PBS (pH 7.4), 0.2% (w / v) pyridoxine hydrochloride, 1% (w / v) BSA, 5% (w / v) trehalose, 0.2% (w / v) Tween-20, and 0.1% (w / v) Proclin 300.

[0100] When using it, the DHEA-S antigen derivative terpyridine ruthenium label is diluted to 0.9 μg / mL using ruthenium dilution buffer.

[0101] 4.4 DHEA-S calibrators; The DHEA-S calibrator includes dehydroepiandrosterone sulfate calibrator and working dilution; The working diluent comprises the following ingredients: 50 mmol / L MES (pH 6.0), 15% (v / v) bovine serum, 0.5% (v / v) L-proline, 5% (v / v) trehalose, 0.15% (v / v) Tween-20, 5% (v / v) glycerol, and 0.2% (v / v) Proclin 300.

[0102] When using, dilute high-purity DHEA-S with working diluent to prepare a calibrator set containing 8 concentration points (0.1, 0.2, 4, 20, 100, 500, 1000, 1200 µg / dL).

[0103] 4.5 DHEA-S quality control products; The DHEA-S quality control products include dehydroepiandrosterone sulfate quality control products and working diluents; When used, the working diluent comprises the following ingredients: 50 mmol / L MES (pH 6.0), 15% (v / v) bovine serum, 0.5% (v / v) L-proline, 5% (v / v) trehalose, 0.15% (v / v) Tween-20, 5% (v / v) glycerol, and 0.2% (v / v) Proclin 300.

[0104] When using, dilute high-purity DHEA-S with working diluent to prepare quality control samples containing 5µg / dL and 500µg / dL.

[0105] 4.6 Concentrated detergent; The washing solution comprises the following ingredients: 1.21% (v / v) trisodium phosphate, 1.53% (v / v) phosphoric acid, 17.25% (v / v) sodium chloride, 1.5% (v / v) Tween-20, and 0.2% (v / v) Proclin 300.

[0106] When using the concentrated washing solution, it should be diluted 20 times with purified water.

[0107] 4.7 Activation solution: The activation solution comprises the following raw materials: 0.1 mol / L piperazine-N,N'-bis(ethanesulfonic acid), 0.1 mol / L tripropylamine, 0.15 mol / L sodium chloride, 0.1% (v / v) Triton X-100 and 0.05% (w / v) Proclin 300.

[0108] When using, add 150 μL to each well immediately after all incubation and washing steps and before instrument testing.

[0109] 4.8 Assembly The streptavidin magnetic bead reagent, biotin-labeled dehydroepiandrosterone sulfate antibody reagent, ruthenium tripyridine-labeled dehydroepiandrosterone sulfate derivative reagent, DHEA-S calibrator, DHEA-S quality control sample, concentrated washing solution, and activation solution are packaged separately into the kit and stored at 2℃~8℃ for later use. The optimal working solution concentration may vary depending on the project.

[0110] Comparative Example 1:

[0111] The difference from Example 1 is that the 0.1 mol / L piperazine-N,N'-bis(ethanesulfonic acid) in the activating solution formulation is replaced with 0.1 mol / L trisodium phosphate.

[0112] Comparative Example 2:

[0113] The difference from Example 1 is that methyl-β-cyclodextrin is not added when preparing the magnetic bead diluent.

[0114] Comparative Example 3:

[0115] The difference from Example 1 is that pyridoxine hydrochloride is not added when preparing the ruthenium diluent.

[0116] Comparative Example 4:

[0117] The difference from Example 1 is that L-proline is not added to the working diluent when preparing the quality control and calibrators.

[0118] Comparative Example 5:

[0119] The difference from Example 1 is that L-proline and trehalose are not added to the working diluent when preparing the quality control and calibrators.

[0120] Application example:

[0121] The instructions for using the reagent kit are as follows: The appropriate sample is human serum or plasma (EDTA anticoagulated). Avoid using samples with hemolysis, lipemia, or severe jaundice. The sample testing steps are as follows: Step 1: Sample addition and first incubation Antibody capture antigen: Add 65 μL of biotin-labeled dehydroepiandrosterone sulfate antibody reagent (0.8 μg / mL), 15 μL of sample, 15 μL of calibrator, and 15 μL of quality control to each reaction vessel in sequence, and incubate at 37°C in the dark for 9 minutes. In this step, if the sample contains DHEA-S antigen, a biotin-labeled dehydroepiandrosterone sulfate antibody-DHEA-S antigen complex will be formed. Step 2: Add ruthenium-labeled antigen, streptavidin magnetic beads, and perform a second incubation. Ruthenium-labeled antigen: Add 65 μL of diluted DHEA-S antigen derivative terpyridine ruthenium-labeled working solution (0.9 μg / mL) to each well to compete with the DHEA-S antigen in the sample for dehydroepiandrosterone sulfate antibody; Add magnetic beads: Add diluted streptavidin magnetic beads (0.8 mg / ml) to each well; Second incubation: Incubate at 37°C in the dark for 9 minutes; in this step, the labeled antibody binds to the antigen captured on the magnetic beads to form a streptavidin magnetic bead-biotin-labeled dehydroepiandrosterone sulfate antibody-DHEA-S antigen complex. Step 3: Washing and Testing Place the reaction vessel on the magnetic separator and let it stand for 2 minutes to allow the magnetic beads to be completely adsorbed to the bottom sidewall of the well; remove and discard the supernatant, avoiding contact with the magnetic beads.

[0122] Add washing buffer: Add 300 μL of washing buffer to each well, let stand for 30 seconds, then discard the buffer. Repeat the washing process 3 times to thoroughly remove unbound free labeled antibody. The cleanliness of this step directly determines the background signal level. Add excitation solution: Immediately add 150 μL of the prepared electrochemiluminescence excitation solution to each well; this step is the starting point for detection, and readings should be taken as soon as possible after adding the solution.

[0123] Step 4: Electrochemiluminescence analyzer detection The reaction vessel was placed in a preheated electrochemiluminescence analyzer for detection, and the light signal value of each well was recorded.

[0124] Performance testing: The test kit was tested according to the US CLSI series documents (such as EP5-A3, EP6-A, EP7-A2, EP17-A2, etc.) and relevant industry standards. The results are as follows: 1. Linear range: Linear range detection of the detection kit prepared in Example 1: Within the range of 0.1-1000 µg / dL, seven concentration points were selected: 0.1 µg / dL, 0.2 µg / dL, 4 µg / dL, 20 µg / dL, 100 µg / dL, 500 µg / dL, and 1000 µg / dL. Each concentration point was measured three times. The mean value was used, and linear regression was performed with the theoretical concentration as the X-axis and the measured concentration as the Y-axis. The linear range curve is shown below. Figure 1 As shown.

[0125] according to Figure 1 The results show that the detection signal and concentration are linearly related, with a linear correlation coefficient (r) ≥ 0.9900. The deviation between each measured concentration point and the theoretical value is within ±10%.

[0126] 2. Accuracy: Accuracy testing of the test kit prepared in Example 1: A low-value human serum sample of known concentration was taken, and a certain volume of high-concentration DHEA-S standard solution was added to prepare samples with theoretical concentrations of 5 μg / dL and 500 μg / dL. The actual concentration of each sample was tested using the test kit prepared in Example 1. The deviation between the actual concentration and the theoretical concentration was within 10%.

[0127] Table 1 Accuracy test results

[0128] 3. Sensitivity (Lowest Detection Limit): 3.1 Sensitivity testing of the detection kit prepared in Example 1: Using a "zero concentration" calibrator (or a matrix confirmed to be DHEA-S-free) as a sample, the test was repeated 20 times, and the mean (X) and standard deviation (SD) of these 20 results were calculated. The LoD of the kit prepared in Example 1 was 0.05 µg / dL (95% confidence interval).

[0129] Table 2. Sensitivity test results of the detection kit prepared in Example 1

[0130] The reagent kit prepared in this application has a limit of detection (LoD) of up to 0.05 µg / dL, which can accurately detect DHEA-S at normal low levels and pathologically reduced levels, meeting the stringent requirements of pediatrics, endocrinology and other departments for low-concentration detection.

[0131] 3.2 Sensitivity test of the detection kit prepared in Comparative Example 2: Using "zero concentration" calibrators (or matrices confirmed to be DHEA-S-free) as samples, the tests were repeated 20 times, and the mean (X) and standard deviation (SD) of these 20 results were calculated. The LoD of the kit prepared in Comparative Example 2 was 0.06 µg / dL, and the LoD verification positive rate was 85%, which did not meet the 95% confidence interval.

[0132] Table 3. Sensitivity test results of the detection kit prepared in Comparative Example 2

[0133] 4. Specificity (cross-reactivity and interference): Specificity detection of the test kit prepared in Example 1: High concentrations of potentially cross-reactive substances, such as DHEA, androstenedione, testosterone, cortisol, and progesterone, were added to the matrix and the cross-reactivity rate of the major analogs was detected using the kit.

[0134] Table 4 Specific detection results

[0135] The test results show that the kit prepared in this application, using uniquely characterized high-affinity and high-specificity antibodies, reduces the cross-reactivity rate of the major structural analog DHEA to <1.0%, far lower than that of conventional kits (usually >5%). The cross-reactivity rates with other common endocrine hormones (such as testosterone and cortisol) are all below 0.3%, fundamentally avoiding false positive or false negative results caused by cross-reactivity and ensuring the specificity and diagnostic accuracy of the test results.

[0136] 5. Intra-batch precision Precision testing of the test kit prepared in Example 1: Precision reference samples of low, medium and high concentrations were selected and tested repeatedly 20 times within the same batch. The test results are shown in the table, indicating that the coefficients of variation of the test kit of this application at low, medium and high concentrations are all <5%.

[0137] Table 5 Precision test results

[0138] 6. Thermal stability: 6.1 Thermal stability test of the test kit prepared in Example 1: The reagent kit was placed at 37°C for 7 and 14 days, and the change in its signal value was tested. The results are shown in the table below. Compared with the control sample stored at 4°C, the signal value of the reagent kit prepared in Example 1 of this application decreased by less than 15% after being placed at 37°C for 7 and 14 days. Other key performance indicators (precision, accuracy, linear range, analytical sensitivity, etc.) also met the above standards.

[0139] Table 6. Thermal stability test results of the test kit prepared in Example 1

[0140] 6.2 Thermal stability test of the test kit prepared in Comparative Example 1: The reagent kit prepared in Comparative Example 1 was placed at 37°C for 7 and 14 days, and the change in its signal value was tested. The results are shown in the table below.

[0141] Table 7. Results of thermal stability testing of the test kit prepared in Comparative Example 1

[0142] As can be seen from Tables 6 and 7, the thermal stability test results of the test kit prepared in Example 1 are better than those of Comparative Example 1, indicating that the improvement of the excitation solution formulation and the addition of piperazine-N,N'-bis(ethanesulfonic acid) buffer in this application provide a more stable coordination environment for ruthenium tripyridine.

[0143] 6.3 Thermal stability test of the test kit prepared in Comparative Example 3: The reagent kit prepared in Comparative Example 3 was placed at 37°C for 7 and 14 days, and the change in its signal value was tested. The results are shown in the table below.

[0144] Table 8. Results of thermal stability testing of the test kit prepared in Comparative Example 3

[0145] As shown in Tables 6 and 8, the thermal stability test results of the test kit prepared in Example 1 are better than those of Comparative Example 3. This indicates that by improving the ruthenium diluent formulation in this application, pyridoxine hydrochloride was introduced as a specific free radical scavenger. The added pyridoxine hydrochloride can not only effectively quench singlet oxygen, but also form a weak complex with the metal ions in the formulation, preventing the degradation of the marker caused by metal ion catalysis. Compared with the conventional formulation, the thermal stability of the reagent is effectively improved and the shelf life of the kit is extended.

[0146] 6.4 Thermal stability test of the test kit prepared in Comparative Example 4: The reagent kit prepared in Comparative Example 4 was placed at 37°C for 7 and 14 days, and the change in its signal value was tested. The results are shown in the table below.

[0147] Table 9. Results of thermal stability tests on the test kits prepared in Comparative Example 4

[0148] As shown in Tables 6 and 9, the thermostability test results of the test kit prepared in Example 1 are better than those of Comparative Example 4. This indicates that by improving the working diluent formulation in this application and using a complex of L-proline and trehalose, L-proline, as an osmotic pressure compensating solute, can more effectively maintain the native conformation of the antibody in the liquid environment through preferential repulsion. In synergy with trehalose, it effectively improves the stability of calibrators and quality control products.

[0149] 6.5 Thermal stability test of the test kit prepared in Comparative Example 5: The reagent kit prepared in Comparative Example 5 was placed at 37°C for 7 and 14 days, and the change in its signal value was tested. The results are shown in the table below.

[0150] Table 10. Results of thermal stability testing of the test kit prepared in Comparative Example 5

[0151] As shown in Tables 6 and 10, the thermostability test results of the test kit prepared in Example 1 are better than those of Comparative Example 5. This indicates that by improving the working diluent formulation in this application, a complex of L-proline and trehalose is used in this formulation. L-proline, as an osmotic pressure compensating solute, can more effectively maintain the native conformation of the antibody in the liquid environment through preferential repulsion. It works synergistically with trehalose to effectively improve the stability of calibrators and quality control products.

[0152] 7. Correlation between clinical serum and Roche reagents The clinical correlation with Roche reagents (n=50) was determined as follows: 50 clinical specimens covering high, medium and low concentrations were collected, and the correlation with Roche reagents was 0.9988.

[0153] The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate prepared in this application has good linear range, accuracy, sensitivity, specificity, precision and thermal stability. This application exhibits extremely low cross-reactivity: employing uniquely characterized high-affinity, high-specificity antibodies, the cross-reactivity rate with the major structural analog DHEA is reduced to <1.0% (e.g., 0.8%), far lower than that of conventional kits (typically >5%). Cross-reactivity rates with other common endocrine hormones (such as testosterone and cortisol) are all below 0.3%, fundamentally avoiding false positive or false negative results caused by cross-reactivity and ensuring the specificity and diagnostic accuracy of the test results.

[0154] The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate prepared in this application has high detection sensitivity: thanks to the signal amplification process of streptavidin and biotin, the high-efficiency ruthenium tripyridine labeling system and the optimized signal amplification technology, the kit prepared in this application has a limit of detection (LoD) of up to 0.05 µg / dL, which can accurately detect low normal values ​​and pathologically reduced levels of DHEA-S, meeting the stringent requirements of pediatrics, endocrinology and other departments for low concentration detection.

[0155] The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate prepared in this application has a broad linear range: the linear correlation coefficient r ≥ 0.9900 within the concentration range of 0.1 µg / dL to 1000 µg / dL. The deviation between the measured and theoretical values ​​at each concentration point is less than ±10%, and a single calibration can cover all clinically relevant concentrations from children to adults, from low to abnormally high levels, without the need for dilution and retesting, greatly simplifying the operation process.

[0156] The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate prepared in this application has long-term reagent stability: by improving the formulation of the diluent, it can be stably stored at 2-8℃ for at least 12 months, and after 7 days of accelerated destructive testing at 37℃, there are no significant changes in various performance indicators.

[0157] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0158] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate, characterized in that: The kit includes: Streptavidin magnetic bead reagent; Biotin-labeled dehydroepiandrosterone sulfate antibody reagent; Tripyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent; and... Activation solution.

2. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 1, characterized in that: The streptavidin magnetic bead reagent includes streptavidin magnetic beads and a magnetic bead diluent; The magnetic bead diluent comprises the following raw materials: neutral buffer 1, methyl-β-cyclodextrin, stabilizer 1, surfactant 1, and preservative 1; Preferably, the neutral buffer 1 comprises at least one of HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 1 is 7-7.5; Preferably, the stabilizer 1 includes at least one of BSA, casein, and fish gelatin; Preferably, the surfactant 1 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 1 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the magnetic bead diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 1% (w / v)-3% (w / v) methyl-β-cyclodextrin, 0.1% (w / v)-1% (w / v) BSA, 0.1% (w / v)-1% (w / v) Tween-20, and 0.05% (w / v)-0.15% (w / v) Proclin 300.

3. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 1, characterized in that: The ruthenium-labeled terpyridine sulfate derivative reagent includes a DHEA-S antigen derivative terpyridine ruthenium label and a ruthenium diluent; The ruthenium diluent comprises the following raw materials: neutral buffer 2, pyridoxine hydrochloride, stabilizer 2, sugar protectant, surfactant 2, and preservative 2; Preferably, the neutral buffer 2 includes one or more of HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 2 is 7-7.5; Preferably, the stabilizer 2 includes at least one of BSA, casein, and fish gelatin; Preferably, the sugar protectant includes at least one of sucrose, trehalose, and dextran; Preferably, the surfactant 2 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 2 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the ruthenium diluent comprises the following ingredients: 5 mmol / L-15 mmol / L PBS, 0.1% (w / v)-0.5% (w / v) pyridoxine hydrochloride, 0.5% (w / v)-1.5% (w / v) BSA, 3% (w / v)-7% (w / v) trehalose, 0.05% (w / v)-0.3% (w / v) Tween-20, and 0.05% (w / v)-0.3% (w / v) Proclin 300.

4. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 1, characterized in that: The activation solution comprises the following raw materials: neutral buffer solution 3, pH adjustment aid, salt substance, surfactant 3, and preservative 3; Preferably, the neutral buffer 3 is a piperazine-N,N'-bis(ethanesulfonic acid) buffer, and the pH of the neutral buffer 3 is 7-7.5; Preferably, the pH adjusting agent is tripropylamine; Preferably, the salt substance includes at least one of sodium chloride, potassium chloride, and magnesium chloride; Preferably, the surfactant 3 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 3 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the activation solution comprises the following raw materials: 0.05 mol / L-0.2 mol / L piperazine-N,N'-bis(ethanesulfonic acid), 0.05 mol / L-0.2 mol / L tripropylamine, 0.1 mol / L-0.2 mol / L sodium chloride, 0.05% (v / v)-0.15% (v / v) Triton X-100, and 0.01% (w / v)-0.1% (w / v) Proclin 300.

5. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 1, characterized in that: The kit also includes calibrators, quality control products, and washing solution.

6. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 5, characterized in that: The calibrators include dehydroepiandrosterone sulfate calibrators and working diluents; the quality control materials include dehydroepiandrosterone sulfate quality control materials and working diluents. The working diluent comprises the following ingredients: neutral buffer 4, protein stabilizer, L-proline, trehalose, surfactant 4, and preservative 4. Preferably, the neutral buffer 4 includes one or more of MES buffer, HEPES buffer, PBS buffer, and Tris buffer; the pH of the neutral buffer 4 is 6-6.

5. Preferably, the protein stabilizer includes at least one of glycerol, bovine serum, and polyethylene glycol; Preferably, the surfactant 4 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 4 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10, and MIT; The working diluent comprises the following ingredients: 40 mmol / L-60 mmol / L MES, 10% (v / v)-20% (v / v) bovine serum, 0.1% (v / v)-1% (v / v) L-proline, 1% (v / v)-10% (v / v) trehalose, 0.05% (v / v)-0.3% (v / v) Tween-20, 1% (v / v)-10% (v / v) glycerol, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

7. The high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 5, characterized in that: The washing liquid comprises the following raw materials: trisodium phosphate, phosphoric acid, salt substances, surfactant 5, and preservative 5; Preferably, the salt substance includes at least one of sodium chloride, potassium chloride, and magnesium chloride; Preferably, the surfactant 5 includes at least one of SDS, CHAPS, CTAB, Tween-20, and Triton X-100; Preferably, the preservative 5 includes at least one of Proclin 300, Proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the washing solution comprises the following raw materials: 1% (v / v)-2% (v / v) trisodium phosphate, 1% (v / v)-2% (v / v) phosphoric acid, 15% (v / v)-20% (v / v) sodium chloride, 1% (v / v)-2% (v / v) Tween-20, and 0.1% (v / v)-0.5% (v / v) Proclin 300.

8. A method for preparing a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate, characterized in that: Prepare kits by providing streptavidin magnetic bead reagent, biotin-labeled dehydroepiandrosterone sulfate antibody reagent, ruthenium tripyridine-labeled dehydroepiandrosterone sulfate derivative reagent, calibrators, quality control products, washing solutions, and activation solutions.

9. The preparation method of a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 8, characterized in that: The biotin-labeled dehydroepiandrosterone sulfate antibody reagent includes a biotin-labeled dehydroepiandrosterone sulfate antibody. The preparation method of the biotin-labeled dehydroepiandrosterone sulfate antibody includes the following steps: DHEA-S monoclonal antibody and biotin are coupled together, and then a blocking agent is added to carry out the reaction to obtain the biotin-labeled dehydroepiandrosterone sulfate antibody. Preferably, the molar ratio of the DHEA-S monoclonal antibody to biotin is 1:(5-20). Preferably, the amount of the sealing agent added is 40-60 μL.

10. The preparation method of a high-sensitivity electrochemiluminescence immunoassay kit for dehydroepiandrosterone sulfate according to claim 8, characterized in that: The terpyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent includes a terpyridine-ruthenium-labeled dehydroepiandrosterone sulfate derivative and a ruthenium diluent; The preparation method of the terpyridine ruthenium-labeled dehydroepiandrosterone sulfate derivative is as follows: the DHEA-S-BSA conjugate is mixed with EDC to obtain the activated DHEA-S-BSA conjugate with a final concentration of 5 mmol / L-20 mmol / L. According to the molar ratio of ruthenium to protein (10-30:1), NHS-Ru(bpy)3 2+ The solution was added to the activated DHEA-S-BSA conjugate to carry out the coupling reaction, and then a blocking agent was added to carry out the reaction to obtain a terpyridine ruthenium-labeled dehydroepiandrosterone sulfate derivative. Preferably, the amount of the sealing agent added is 40-60 μL.

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