CN-121978355-A - Sparaglutide electrochemiluminescence immunoassay method and kit based on TSA signal amplification technology

Abstract

The present invention relates to an electrochemiluminescence immunoassay method and reagent kit for detecting semaglutide based on TSA signal amplification technology, which solves the technical problem of how to detect semaglutide with simple operation, fast, wide detection range, and high detection sensitivity. It combines electrochemiluminescence immunoassay with TSA signal amplification to achieve simple and fast detection of semaglutide with wide detection range and high detection sensitivity.

Inventors

• ZOU LINGLONG
• Liu Qianrui
• ZHANG HEFENG
• ZOU CHANGHONG
• LUO YIFAN

Assignees

• 温州康瑞佰欧生物技术有限公司

Dates

Publication Date

20260505

Application Date

20260114

Claims (8)

1. 1. The method for detecting the electrochemical luminescence immunity of the semaglutin based on the TSA signal amplification technology is characterized by comprising the following steps of: first, preparation of HRP-labeled detection antibody: Performing HRP labeling on the semaglutin antibody to obtain an HRP labeled detection antibody reagent; Secondly, preparing ruthenium-labeled streptavidin: Ruthenium labeling is carried out on streptavidin to obtain ruthenium labeled streptavidin; Thirdly, adding a coating solution of the semaglutin capture antibody into the electrochemical luminescence micro-pore plate, incubating, adding a washing buffer solution into the electrochemical luminescence micro-pore plate for washing, adding a semaglutin sample, incubating, washing the plate, and then adding the HRP-labeled detection antibody reagent obtained in the first step into the electrochemical luminescence micro-pore plate for washing after incubation; Fourth, biotin-tyramide is deposited in situ based on TSA signal amplification technique: Adding a biotin-tyramide and H 2 O 2 mixed solution into an electrochemiluminescence microplate, and amplifying TSA signals; fifth, ruthenium labeled streptavidin is combined with biotin: adding ruthenium label streptavidin into the electrochemiluminescence micro-pore plate, incubating and washing the plate; step six, quantitatively detecting the semaglutin by using an electrochemiluminescence immunoassay analyzer: And adding an electrochemiluminescence substrate solution into the electrochemiluminescence micro-pore plate, and reading an electrochemiluminescence value.
2. 2. The method for electrochemical luminescence immunoassay of semaglutin based on TSA signal amplification technology according to claim 1, wherein the concentration of the semaglutin capture antibody coating liquid is 1-10 μg/mL, and the concentration of the HRP-labeled detection antibody reagent is 0.5-4 μg/mL.
3. 3. The TSA signal amplification technology-based semaglutin electrochemiluminescence immunoassay method of claim 2, wherein the concentration of the semaglutin capture antibody coating solution is 10 μg/mL, and the concentration of the HRP-labeled detection antibody reagent is 2 μg/mL.
4. 4. The TSA signal amplification technology-based semaglutin electrochemiluminescence immunoassay method of claim 1, wherein in the first step, the semaglutin antibody is labeled using HRP labeling kit.
5. 5. The TSA signal amplification technology-based semaglutin electrochemiluminescence immunoassay according to claim 1, wherein in the second step, streptavidin is labeled using a ruthenium label kit.
6. 6. The TSA signal amplification technology-based semaglutin electrochemiluminescence immunoassay method of claim 1, wherein the electrochemiluminescence substrate solution is a coreactant tri-n-propylamine.
7. 7. The method for electrochemical luminescence immunoassay of semaglutin based on TSA signal amplification technology according to claim 1, wherein the time of the TSA signal amplification process is 5-10 min.
8. 8. The kit is characterized by comprising an HRP-labeled semaglutin detection antibody reagent, ruthenium-labeled streptavidin, semaglutin capture antibody coating liquid and a biotin-tyramide mixed solution with H 2 O 2 .

Description

Sparaglutide electrochemiluminescence immunoassay method and kit based on TSA signal amplification technology Technical Field The invention relates to the technical field of polypeptide drug concentration detection, in particular to a semaglutin electrochemiluminescence immunoassay method and a kit based on a TSA signal amplification technology. Background Semaglutin (Semaglutide) is a novel long-acting glucagon-like peptide-1 (GLP-1) receptor agonist and is widely applied to the treatment of type 2 diabetes and obesity. With the explosive growth of clinical application, accurate and sensitive quantitative detection of semaglutin in biological samples (such as serum and plasma) is increasingly demanded in the fields of clinical Treatment Drug Monitoring (TDM), pharmacokinetic (PK) research, forensic toxicology identification, and anti-contrastimulant detection. However, since semaglutinin itself is a peptide drug with a relatively large molecular weight (about 4113 Da), complex modifications, and extremely low blood concentration (typically at nanograms per milliliter to picograms per milliliter level) at therapeutic doses, its detection faces significant technical challenges. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is currently considered to be the "gold standard" for quantitative detection of semaglutin. However, LC-MS/MS detection procedures are complex and there are a number of key bottlenecks such as complex sample pretreatment, low detection throughput, large sample loss, etc. Meanwhile, the conventional immunoassay (ELISA, electrochemiluminescence immunoassay and the like) is adopted to detect the semaglutin, so that the problems of insufficient sensitivity and the like exist. Therefore, development of a method for detecting semaglutin, which is simple and rapid to operate, wide in detection range and high in detection sensitivity, is needed. Disclosure of Invention The application provides a simple, convenient and high-sensitivity electrochemical luminescence immunoassay method and a kit for detecting semaglutin based on a TSA signal amplification technology, which aim to solve the technical problems of how to detect semaglutin simply, rapidly, widely in detection range and high in detection sensitivity. In a first aspect of the present disclosure, there is provided a semaglutin electrochemiluminescence immunoassay method based on TSA signal amplification technology, comprising the steps of: first, preparation of HRP-labeled detection antibody: Performing HRP labeling on the semaglutin antibody to obtain an HRP labeled detection antibody reagent; Secondly, preparing ruthenium-labeled streptavidin: Ruthenium labeling is carried out on streptavidin to obtain ruthenium labeled streptavidin; Thirdly, adding a coating solution of a semaglutin capture antibody into the electrochemical luminescence micro-pore plate, incubating, adding a washing buffer solution into the electrochemical luminescence micro-pore plate for washing, adding a semaglutin sample, incubating, washing the plate, adding the HRP-labeled detection antibody reagent obtained in the first step into the electrochemical luminescence micro-pore plate, and washing after incubation; Fourth, biotin-tyramide is deposited in situ based on TSA signal amplification technique: Adding a biotin-tyramide and H 2O2 mixed solution into an electrochemiluminescence microplate, and amplifying TSA signals; fifth, ruthenium labeled streptavidin is combined with biotin: adding ruthenium label streptavidin into the electrochemiluminescence micro-pore plate, incubating and washing the plate; step six, quantitatively detecting the semaglutin by using an electrochemiluminescence immunoassay analyzer: And adding an electrochemiluminescence substrate solution into the electrochemiluminescence micro-pore plate, and reading an electrochemiluminescence value. Preferably, the concentration of the coating liquid of the semaglutin capture antibody is 1-10 mug/mL, and the concentration of the HRP-labeled detection antibody reagent is 0.5-4 mug/mL. Further preferably, the concentration of the coating solution of the semaglutin capture antibody is 10 μg/mL, and the concentration of the HRP-labeled detection antibody reagent is 2 μg/mL. Preferably, in the first step, the semaglutin antibody is labeled using an HRP labeling kit. Preferably, in the second step, streptavidin is labeled using a ruthenium labeling kit. Preferably, the electrochemiluminescence substrate solution is tri-n-propylamine as co-reactant. Preferably, in the fourth step, the time of the TSA signal amplification processing process is 5-10 min. In a second aspect of the invention, a kit is provided, comprising an HRP-labeled semaglutin detection antibody reagent, ruthenium-labeled streptavidin, semaglutin capture antibody coating solution, and biotin-tyramide mixed solution with H 2O2. The tyramide signal amplification Technology (TSA) has the advantages of simple method, high sensitivity an