CN-122017259-A - High-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit and preparation method thereof

CN122017259ACN 122017259 ACN122017259 ACN 122017259ACN-122017259-A

Abstract

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

Inventors

DU YUANYUAN
ZHANG SONG
TONG CHAO

Assignees

厦门半上生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260210

Claims (10)

1. A high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit is characterized by comprising the following components: streptavidin magnetic bead reagent; Biotin-labeled dehydroepiandrosterone sulfate antibody reagent; dehydroepiandrosterone sulfate derivative reagent marked by terpyridyl ruthenium and preparation method thereof Excitation liquid.
2. The high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay kit according to claim 1, wherein the streptavidin magnetic bead reagent comprises streptavidin magnetic beads and magnetic bead diluent; wherein the magnetic bead diluent comprises the following raw materials of neutral buffer solution 1, methyl-beta-cyclodextrin, stabilizer 1, surfactant 1 and preservative 1; preferably, the neutral buffer solution 1 comprises at least one of HEPES buffer solution, PBS buffer solution and Tris buffer solution, wherein the pH value of the neutral buffer solution 1 is 7-7.5; preferably, the stabilizer 1 comprises at least one of BSA, casein and fish gelatin; Preferably, the surfactant 1 comprises at least one of SDS, CHAPS, CTAB, tween-20 and Triton X-100; Preferably, the preservative 1 comprises at least one of Proclin 300, proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the magnetic bead diluent comprises the following raw materials of 5mmol/L-15mmol/L PBS, 1% (w/v) -3% (w/v) methyl-beta-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 dehydroepiandrosterone sulfate electrochemical luminescence immunoassay detection kit according to claim 1, wherein the terpyridyl ruthenium labeled dehydroepiandrosterone sulfate derivative reagent comprises a DHEA-S antigen derivative terpyridyl ruthenium label and ruthenium diluent; Wherein the ruthenium diluent comprises the following raw materials of neutral buffer solution 2, pyridoxine hydrochloride, stabilizer 2, saccharide protecting agent, surfactant 2 and preservative 2; Preferably, the neutral buffer 2 comprises one or more of HEPES buffer, PBS buffer and Tris buffer, wherein the pH of the neutral buffer 2 is 7-7.5; Preferably, the stabilizer 2 comprises at least one of BSA, casein and fish gelatin; preferably, the saccharide protecting agent includes at least one of sucrose, trehalose and dextran; preferably, the surfactant 2 comprises at least one of SDS, CHAPS, CTAB, tween-20 and Triton X-100; preferably, the preservative 2 comprises at least one of Proclin 300, proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the ruthenium diluent comprises the following raw materials of 5mmol/L-15mmol/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, 0.05% (w/v) -0.3% (w/v) Proclin 300.
4. The high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay kit according to claim 1, wherein the excitation solution comprises neutral buffer solution 3, pH adjusting auxiliary agent, salt substance, surfactant 3 and preservative 3; preferably, the neutral buffer solution 3 is piperazine-N, N' -bis (ethane sulfonic acid) buffer solution, and the pH value of the neutral buffer solution 3 is 7-7.5; Preferably, the pH adjusting auxiliary agent is tripropylamine; Preferably, the salt substance comprises at least one of sodium chloride, potassium chloride and magnesium chloride; preferably, the surfactant 3 comprises at least one of SDS, CHAPS, CTAB, tween-20 and Triton X-100; Preferably, the preservative 3 comprises at least one of Proclin 300, proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the excitation solution comprises the following raw materials of 0.05mol/L-0.2mol/L piperazine-N, N' -bis (ethane sulfonic acid), 0.05mol/L-0.2mol/L tripropylamine, 0.1mol/L-0.2mol/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 dehydroepiandrosterone sulfate electrochemiluminescence immunoassay kit according to claim 1, wherein the kit further comprises a calibrator, a quality control material and a washing solution.
6. The high-sensitivity dehydroepiandrosterone-sulfate electrochemiluminescence immunoassay detection kit according to claim 5, wherein the calibrator comprises a dehydroepiandrosterone-sulfate calibrator and a working diluent, and the quality control comprises a dehydroepiandrosterone-sulfate quality control and a working diluent; wherein the working diluent comprises the following raw materials of neutral buffer solution 4, protein stabilizer, L-proline, trehalose, surfactant 4 and preservative 4, Preferably, the neutral buffer solution 4 comprises one or more of MES buffer solution, HEPES buffer solution, PBS buffer solution and Tris buffer solution, wherein the pH value of the neutral buffer solution 4 is 6-6.5; Preferably, the protein stabilizer comprises at least one of glycerol, bovine serum and polyethylene glycol; preferably, the surfactant 4 comprises at least one of SDS, CHAPS, CTAB, tween-20 and Triton X-100; preferably, the preservative 4 comprises at least one of Proclin 300, proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; The working diluent comprises 40mmol/L-60mmol/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 dehydroepiandrosterone sulfate electrochemiluminescence immunoassay kit according to claim 5, wherein the washing solution comprises trisodium phosphate, phosphoric acid, salt substances, surfactant 5 and preservative 5; Preferably, the salt substance comprises at least one of sodium chloride, potassium chloride and magnesium chloride; Preferably, the surfactant 5 comprises at least one of SDS, CHAPS, CTAB, tween-20 and Triton X-100; Preferably, the preservative 5 comprises at least one of Proclin 300, proclin 950, gentamicin sulfate, BIT-10, BND-10 and MIT; Preferably, the washing liquid comprises 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 preparation method of a high-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit is characterized by respectively providing a streptavidin magnetic bead reagent, a biotin-labeled dehydroepiandrosterone sulfate antibody reagent, a terpyridyl ruthenium-labeled dehydroepiandrosterone sulfate derivative reagent, a calibrator, a quality control product, a washing solution and an excitation solution, and preparing the kit.
9. The method for preparing the high-sensitivity dehydroepiandrosterone-sulfate electrochemical luminescence immunoassay detection kit according to claim 8, wherein the biotin-labeled dehydroepiandrosterone-sulfate antibody reagent comprises a biotin-labeled dehydroepiandrosterone-sulfate antibody; The preparation method of the biotin-labeled dehydroepiandrosterone-sulfate antibody comprises the following steps of performing a coupling reaction on a DHEA-S monoclonal antibody and biotin, and adding a blocking agent for reaction to obtain the biotin-labeled dehydroepiandrosterone-sulfate antibody; Preferably, the mole ratio of the DHEA-S monoclonal antibody to the biotin is 1 (5-20); Preferably, the blocking agent is added in an amount of 40-60. Mu.L.
10. The method for preparing the high-sensitivity dehydroepiandrosterone sulfate electrochemical luminescence immunoassay detection kit according to claim 8, wherein the terpyridyl ruthenium marked dehydroepiandrosterone sulfate derivative reagent comprises a terpyridyl ruthenium marked dehydroepiandrosterone sulfate derivative and a ruthenium diluent; The preparation method of the terpyridyl ruthenium marked dehydroepiandrosterone sulfate derivative comprises the following steps of mixing a DHEA-S-BSA conjugate with EDC to obtain an activated DHEA-S-BSA conjugate with a final concentration of 5mmol/L-20 mmol/L; adding NHS-Ru (bpy) 3 2+ solution into the activated DHEA-S-BSA conjugate according to the mol ratio (10-30) of ruthenium to protein to perform coupling reaction, and adding a blocking agent to perform reaction to obtain the terpyridyl ruthenium marked dehydroepiandrosterone sulfate derivative; Preferably, the blocking agent is added in an amount of 40-60. Mu.L.

Description

High-sensitivity dehydroepiandrosterone sulfate electrochemiluminescence immunoassay detection kit and preparation method thereof Technical Field The application relates to the technical field of biomedical detection, in particular to a high-sensitivity dehydroepiandrosterone-sulfate electrochemiluminescence immunoassay detection kit and a preparation method thereof. Background Dehydroepiandrosterone sulfate (DHEA-S) is the most abundant steroid hormone in the human blood circulation, mainly secreted by the adrenal cortex reticulum. The level detection has important clinical value for evaluating adrenocortical function, diagnosing polycystic ovary syndrome, congenital adrenocortical hyperplasia, cushing's syndrome and some endocrine related tumors. In the biomedical detection field, the immunoassay technology aiming at the target marker DHEA-S is continuously iterated along with the demand, and technical paths such as a radioimmunoassay method, an enzyme-linked immunosorbent assay method, a chemiluminescence method and the like are developed successively, but the prior art still has obvious short plates in the aspects of sensitivity, stability, detection efficiency, industrial suitability and the like, and is difficult to meet the high standard demands of clinical diagnosis and market application, and the specific state of the art and limitation are as follows: 1. Radioimmunoassay The method is used as a classical technology of early-stage immunodetection, and realizes quantitative analysis of antigen-antibody reaction by means of a radioactive marker, but has the fundamental application defects that the detection process is accompanied by radioactive pollution, the requirements on the operation environment, protective equipment and personnel are extremely high, the effective period of the reagent is extremely short due to radioactive attenuation, the reagent needs to be replaced at high frequency to ensure the detection reliability, the operation flow is complicated, the large-scale and standardized detection cannot be realized by automatic equipment, and the overall efficiency is low. 2. Enzyme-linked immunosorbent assay The quantitative detection is completed through enzyme catalytic substrate color development, but the detection performance and the practicability are still obviously insufficient, the sensitivity is low, the linear detection range is narrow, the detection precision of a low-concentration sample is insufficient, the operation steps are redundant, the single detection period is long, and more importantly, the stability of a detection result is easily interfered by factors such as environmental temperature, reaction time deviation and the like, and the precision in and among batches is difficult to guarantee. 3. Existing chemiluminescent methods By virtue of high sensitivity and automatic detection capability, a chemiluminescence method has become a main technical route of the current immunodetection market, but aiming at detection scenes of specific markers such as DHEA-S, the existing chemiluminescence products still have a multi-dimensional improvement space, such as: The sensitivity and specificity are that the cross reaction rate of the detection antibody and the structural analogue is high, the nonspecific binding is easy to be induced, and the detection of a low-concentration sample is inaccurate or the recognition capability of an interfering substance is weak; The reaction stability is that the luminous signal decay rate is high, the effective detection window period is short, the consistency of detection results in different batches and the same batch is difficult to ensure, and the precision is limited; Reagent stability, namely the activity of the core components of the kit, such as enzyme markers and luminous substrates, is easy to decay in the long-term storage or transportation process, and the shelf life and the detection reliability of the reagent are directly affected; The detection speed is that the dynamic process of antigen-antibody reaction is slow, and the total detection time is difficult to match with the requirement of clinical rapid diagnosis. In addition, the DHEA-S chemiluminescent detection reagent in the prior art also adopts an acridinium ester chemiluminescent platform, and because the acridinium ester belongs to a flashing type luminescent system, the luminescent signal capture is highly dependent on a precise instrument, so that the precision is insufficient, and the kit cannot meet the market demand. Therefore, how to effectively solve the problems of insufficient sensitivity and specificity, poor stability, low detection speed and the like in the DHEA-S detection field in the prior art becomes a problem to be solved. Disclosure of Invention Aiming at the defects of the prior art, the application provides a high-sensitivity dehydroepiandrosterone-sulfate electrochemiluminescence immunoassay detection kit and a preparation method thereof. The application d