Smeglutide electrochemiluminescence immunoassay method and kit based on TSA signal amplification technology

Abstract

The invention relates to a semeglutide electrochemiluminescence immunoassay method based on a TSA signal amplification technology and a kit, and solves the technical problem of how to detect semeglutide with simple and rapid operation, wide detection range and high detection sensitivity. The method is simple to operate, can quickly detect the semeglutide, and is wide in detection range and high in detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of peptide drug concentration detection technology, and more specifically, to a method and kit for detecting semaglutide using electrochemiluminescence immunoassay based on TSA signal amplification technology. Background Technology

[0002] Semaglutide is a novel, long-acting glucagon-like peptide-1 (GLP-1) receptor agonist widely used in the treatment of type 2 diabetes and obesity. With its explosive growth in clinical applications, there is an increasing demand for accurate and sensitive quantitative detection of semaglutide in biological samples (such as serum and plasma) in therapeutic drug monitoring (TDM), pharmacokinetic (PK) studies, forensic toxicology identification, and anti-doping testing.

[0003] However, semaglutide is a complexly modified peptide drug with a large molecular weight (approximately 4113 Da), and its blood concentration at therapeutic doses is extremely low (typically at the nanogram / mL to picogram / mL level), posing significant technical challenges to its detection. Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is considered the "gold standard" for the quantitative detection of semaglutide. However, the LC-MS / MS detection process is complex and suffers from several key bottlenecks, such as complex sample pretreatment, low throughput, and significant sample loss. Furthermore, traditional immunoassays (ELISA, electrochemiluminescence immunoassay, etc.) for semaglutide detection suffer from insufficient sensitivity. Therefore, there is an urgent need to develop a simple, rapid, wide-range, and highly sensitive method for the detection of semaglutide. Summary of the Invention

[0004] This application aims to solve the technical problem of how to detect semaglutide in a simple, rapid, wide-range, and highly sensitive manner, and provides a simple, convenient, and highly sensitive electrochemiluminescent immunoassay method and kit for semaglutide based on TSA signal amplification technology.

[0005] The first aspect of this disclosure provides an electrochemiluminescence immunoassay method for detecting semaglutide based on TSA signal amplification technology, comprising the following steps: Step 1: Preparation of HRP-labeled detection antibodies: HRP-labeled semaglutide antibody was used to obtain an HRP-labeled detection antibody reagent. The second step is the preparation of ruthenium-labeled streptavidin: Ruthenium labeling of streptavidin yielded ruthenium-labeled streptavidin. The third step involves adding the smegglutinin capture antibody coating solution to the electrochemiluminescence microplate and incubating it; then adding washing buffer to the electrochemiluminescence microplate for washing; adding the smegglutinin sample, incubating, and washing the plate; next, adding the HRP-labeled detection antibody reagent obtained in the first step to the electrochemiluminescence microplate, incubating, and then washing. Step 4: In-situ deposition of biotin-tyrosamide based on TSA signal amplification technology: A mixed solution of biotin-tyrosamide and H2O2 was added to an electrochemiluminescence microplate, and the signal was amplified by TSA. Step 5: Ruthenium-labeled streptavidin binds to biotin: Add ruthenium-labeled streptavidin to the electrochemiluminescence microplate, incubate, and wash the plate; Step 6: Quantitative detection of smegglutinin was performed using an electrochemiluminescence immunoassay analyzer. Add the electrochemiluminescence substrate solution to the electrochemiluminescence microplate and read the electrochemiluminescence value.

[0006] Preferably, the concentration of the semaglutide capture antibody coating solution is 1–10 μg / mL, and the concentration of the HRP-labeled detection antibody reagent is 0.5–4 μg / mL. More preferably, the concentration of the semaglutide capture antibody coating solution is 10 μg / mL, and the concentration of the HRP-labeled detection antibody reagent is 2 μg / mL.

[0007] Preferably, in the first step, the smegglutinin antibody is labeled using an HRP labeling kit.

[0008] Preferably, in the second step, streptavidin is labeled using a ruthenium labeling kit.

[0009] Preferably, the electrochemiluminescence substrate solution is tri-n-propylamine, a co-reactant.

[0010] Preferably, the fourth step, the TSA signal amplification process, takes 5 to 10 minutes.

[0011] In a second aspect, the present invention provides a kit comprising an HRP-labeled semaglutide detection antibody reagent, a ruthenium-labeled streptavidin, a semaglutide capture antibody coating solution, and a biotin-tyrosamide and H2O2 mixed solution.

[0012] Tyrosine signal amplification (TSA) is popular due to its simple method and high sensitivity.

[0013] This invention combines electrochemiluminescence immunoassay with TSA to construct a semaglutide detection platform. The combination of electrochemiluminescence immunoassay with signal amplification technology improves sensitivity.

[0014] The advantages of this invention are that the method is simple, the detection process is fast, the detection range is wide, the detection sensitivity is high, and the throughput is high.

[0015] The capture antibody was directly modified onto the electrochemiluminescent microplate, forming a double-antibody sandwich structure between the capture antibody, smegglutinin, and HRP-labeled detection antibody. Then, biotin-tyrosamide was deposited in situ onto the surface of the adjacent protein. Finally, a large number of electroactive luminescent probes, ruthenium, were labeled into the system by biotin and ruthenium-labeled streptavidin, thereby improving the detection sensitivity.

[0016] Further features and aspects of this disclosure will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0017] Figure 1 This is a flowchart of the electrochemiluminescence immunoassay based on TSA signal amplification of the present invention; Figure 2 This is a diagram of the TSA in-situ labeling mechanism; Figure 3 This refers to the detection results of a two-dimensional concentration matrix combination of the capture antibody and the detection antibody in Example 2; Figure 4 This is a comparison of the optical signals of the detection results under several different TSA response times in Example 3; Figure 5 This is Example 4, the standard curve established; Figure 6 This is Example 5, specific data. Detailed Implementation

[0018] The specific embodiments described below are merely preferred embodiments of this application, and the scope of protection of this application is not limited thereto. Those skilled in the art can make modifications or variations based on the principles, concepts, and spirit of this application, and the resulting technical solutions should all be covered within the scope of protection of this application.

[0019] Example 1

[0020] refer to Figure 1 The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology includes the following steps: Step 1: Preparation of HRP-labeled detection antibodies: Using CRIVVIN ® The semaglutide antibody was labeled using an HRP labeling kit, and the experimental procedure was strictly followed according to the instructions. A commercially available semaglutide antibody was used. The specific procedure is as follows: Using 0.01 M CB buffer at 4 ℃, centrifuged at 1000×g for 8 min, the ultrafiltration tube was equilibrated and the semaglutide antibody stock solution was replaced sequentially to dilute the antibody to be labeled (semaglutide antibody) to 2.0 mg / mL for later use.

[0021] The antibody to be labeled: pre-activated HRP: labeling initiation solution was mixed in a ratio of 250:100:63 (v / v / v) and shaken at 37 °C in the dark (500 rpm) for 2 h to conjugate the antibody with HRP. Then, 50.0 μL of labeling stop solution was added to the mixture, and the mixture was incubated at 25 °C in the dark for 1 h to terminate the conjugation reaction, yielding the HRP-antibody conjugate solution.

[0022] The HRP-antibody conjugate solution was purified by centrifugation using PBS buffer.

[0023] Finally, add an equal volume of labeling preservation solution, mix well, and store the HRP-labeled detection antibody reagent at 4°C.

[0024] The second step is the preparation of ruthenium-labeled streptavidin: Using CRIVVIN ® A ruthenium-labeled kit was used to label streptavidin. First, a Ru activation solution was prepared by diluting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sodium N-hydroxythiosuccinimide (NHS) to 100.0 mg / mL using 2-morpholine ethanesulfonate buffer (MES); simultaneously, lyophilized Ru powder was prepared to a concentration of 20.0 mg / mL. Subsequently, a Ru activation solution was prepared at a ratio of Ru: NHS: EDC = 50:20:20 (V / V / V), and incubated at 25°C with shaking in the dark for 25 min to activate Ru. The streptavidin to be labeled was then mixed with the Ru activation solution at a 1:1 (mol / mol) ratio and incubated at 25°C with shaking in the dark for 2 h.

[0025] After labeling, the mixed solution can be purified by ultrafiltration.

[0026] The third step is to construct a double-antibody sandwich immune system by introducing a catalyst for TSA. This catalyst can be HRP (horseradish peroxidase) or a peroxidase-like enzyme, preferably HRP (horseradish peroxidase).

[0027] The semaglutide capture antibody was diluted using a coating diluent to obtain a semaglutide capture antibody coating solution. A commercially available semaglutide capture antibody was used.

[0028] Add 100.0 μL of semaglutide capture antibody coating solution to a 96-well electrochemiluminescence microplate and incubate overnight at 4°C. Add 300 μL of PBST working solution to each well, pat dry, and wash 3 times. Then, add 100.0 μL of semaglutide sample to each well and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. Immediately afterward, add the HRP-labeled detection antibody reagent obtained in the first step to the 96-well electrochemiluminescence microplate and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0029] Step 4: In-situ deposition of biotin-tyrosamide based on TSA signal amplification technology: Adding a mixed solution of biotin-tyrosamide and H2O2 to the double-antibody sandwich system obtained in the third step, under the action of the catalyst HRP and after TSA signal amplification, a large amount of biotin is deposited in situ onto the surface of HRP or its adjacent proteins, thereby increasing the amount of ruthenium-labeled streptavidin in the subsequent process.

[0030] The specific principle is as follows: Figure 2 As shown, HRP catalyzes tyrosine molecules in the presence of H2O2, causing them to generate reactive oxygen species (ROS) intermediates. These intermediates have extremely short lifetimes (milliseconds) and can only diffuse over a very small area (approximately 0-5 micrometers). Within this diffusion range, they covalently cross-link with nearby proteins (primarily electron-rich tyrosine residues), thereby labeling biotin into the electrochemiluminescent microplate.

[0031] Step 5: Ruthenium-labeled streptavidin binds to biotin: Add 100.0 μL of ruthenium-labeled streptavidin to the electrochemiluminescence microplate obtained in step 4, and incubate with shaking at 25 °C for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0032] The sixth step involves using an electrochemiluminescence immunoassay analyzer to quantitatively detect smegglutinin.

[0033] Add 100.0 μL of tri-n-propylamine read buffer to the electrochemiluminescence microplate and read the electrochemiluminescence value at a wavelength of 620 nm.

[0034] Example 2

[0035] To determine the optimal antibody concentration for the detection system, a checkerboard method was used to optimize the capture and detection antibodies. Different concentrations of capture and detection antibodies were combined in a matrix using the checkerboard method, and the optimal concentrations were selected by analyzing the signal-to-noise ratio. The specific process is as follows: Step (1): Prepare HRP-labeled detection antibody.

[0036] Using CRIVVIN ® The HRP labeling kit was used to label the semaglutide antibody, and the experimental procedure was strictly followed according to the instructions. The specific procedure is as follows: Using 0.01 M CB buffer at 4 ℃, centrifuged at 1000×g for 8 min, the ultrafiltration tube was equilibrated and the semaglutide antibody stock solution was replaced sequentially to dilute the antibody to be labeled (semaglutide antibody) to 2.0 mg / mL for later use.

[0037] The antibody to be labeled: pre-activated HRP: labeling initiation solution was mixed in a ratio of 250:100:63 (v / v / v) and shaken at 37 °C in the dark (500 rpm) for 2 h to conjugate the antibody with HRP. Then, 50.0 μL of labeling stop solution was added to the mixture, and the mixture was incubated at 25 °C in the dark for 1 h to terminate the conjugation reaction, yielding the HRP-antibody conjugate solution.

[0038] The HRP-antibody conjugate solution was purified by centrifugation using PBS buffer.

[0039] Finally, add an equal volume of labeling preservation solution, mix well, and store the HRP-labeled detection antibody at 4°C.

[0040] Step (2): Prepare ruthenium-labeled streptavidin.

[0041] Using CRIVVIN ® A ruthenium-labeled kit was used to label streptavidin. First, a Ru activation solution was prepared by diluting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sodium N-hydroxythiosuccinimide (NHS) to 100.0 mg / mL using 2-morpholine ethanesulfonate buffer (MES); simultaneously, lyophilized Ru powder was prepared to a concentration of 20.0 mg / mL. Subsequently, a Ru activation solution was prepared at a ratio of Ru:NHS:EDC = 50:20:20 (V / V / V), and incubated at 25°C with shaking in the dark for 25 min to activate Ru. The streptavidin to be labeled was then mixed with the Ru activation solution at a 1:1 (mol / mol) ratio and incubated at 25°C with shaking in the dark for 2 h.

[0042] After labeling, the mixed solution can be purified by ultrafiltration.

[0043] Step (3): Construct a double-antibody sandwich immune system by introducing the TSA catalyst HRP.

[0044] Add 100.0 μL of semaglutide capture antibody coating solution of different concentrations (1 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL) to a 96-well electrochemiluminescence microplate, and incubate overnight at 4°C. Add 300 μL of PBST working solution to each well, pat dry, and wash 3 times. Then, add 0.1 ng / mL semaglutide sample, 100.0 μL per well, and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. Then, add the HRP-labeled detection antibody reagent obtained in step (1) at different concentrations (0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL) to the 96-well electrochemiluminescence microplate, and incubate with shaking at 25°C for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. μL / well, wash 3 times.

[0045] Step (4): Biotin-tyrosamide is deposited in situ based on TSA signal amplification technology.

[0046] Add a mixture of biotin-tyrosamide and H2O2 to the double-antibody sandwich system obtained in step (3). After reacting for a period of time under the action of catalyst HRP, a large amount of biotin is deposited in situ onto the surface of HRP or its adjacent proteins after TSA signal amplification, thereby increasing the amount of subsequent ruthenium-labeled streptavidin.

[0047] Step (5): Ruthenium-labeled streptavidin binds to biotin.

[0048] Add 100.0 μL of ruthenium-labeled streptavidin to the electrochemiluminescence microplate after the fourth step of treatment, and incubate with shaking at 25 °C for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0049] Step (6): Use an electrochemiluminescence immunoassay analyzer to quantitatively detect smegglutinin.

[0050] Tri-n-propylamine read buffer (100.0 μL / well) was added to the electrochemiluminescence microplate as a co-reactant, and the electrochemiluminescence values ​​were read at a wavelength of 620 nm. The data are as follows: Figure 3 As shown, the highest signal-to-noise ratio (S / N) was observed when the semaglutide capture antibody coating solution was 10 μg / mL and the HRP-labeled detection antibody was 2 μg / mL. Therefore, the optimal combination is 10 μg / mL capture antibody and 2 μg / mL detection antibody.

[0051] Example 3

[0052] TSA reaction time optimization.

[0053] Step (1): Prepare HRP-labeled detection antibody.

[0054] Using CRIVVIN ® The HRP labeling kit was used to label the semaglutide antibody, and the experimental procedure was strictly followed according to the instructions. The specific procedure is as follows: Using 0.01 M CB buffer at 4 ℃, centrifuged at 1000×g for 8 min, the ultrafiltration tube was equilibrated and the semaglutide antibody stock solution was replaced sequentially to dilute the antibody to be labeled (semaglutide antibody) to 2.0 mg / mL for later use.

[0055] The antibody to be labeled: pre-activated HRP: labeling initiation solution was mixed in a ratio of 250:100:63 (v / v / v) and shaken at 37 °C in the dark (500 rpm) for 2 h to conjugate the antibody with HRP. Then, 50.0 μL of labeling stop solution was added to the mixture, and the mixture was incubated at 25 °C in the dark for 1 h to terminate the conjugation reaction, yielding the HRP-antibody conjugate solution.

[0056] The HRP-antibody conjugate solution was purified by centrifugation using PBS buffer.

[0057] Finally, add an equal volume of labeling preservation solution, mix well, and store the HRP-labeled detection antibody at 4°C.

[0058] Step (2): Prepare ruthenium-labeled streptavidin.

[0059] Using CRIVVIN ® A ruthenium-labeled kit was used to label streptavidin. First, a Ru activation solution was prepared by diluting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sodium N-hydroxythiosuccinimide (NHS) to 100.0 mg / mL using 2-morpholine ethanesulfonate buffer (MES); simultaneously, lyophilized Ru powder was prepared to a concentration of 20.0 mg / mL. Subsequently, a Ru activation solution was prepared at a ratio of Ru:NHS:EDC = 50:20:20 (V / V / V), and incubated at 25°C with shaking in the dark for 25 min to activate Ru. The streptavidin to be labeled was then mixed with the Ru activation solution at a 1:1 (mol / mol) ratio and incubated at 25°C with shaking in the dark for 2 h.

[0060] After labeling, the mixed solution can be purified by ultrafiltration.

[0061] Step (3): Construct a double-antibody sandwich immune system by introducing the TSA catalyst HRP.

[0062] Add 10 μg / mL, 100.0 μL of semaglutide capture antibody coating solution to a 96-well electrochemiluminescence microplate and incubate overnight at 4°C. Add 300 μL of PBST working solution to each well, pat dry, and wash 3 times. Then, add 0.1 ng / mL, 00.0 μL of semaglutide sample and incubate at 25°C with shaking for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. Immediately add 100.0 μL of the HRP-labeled detection antibody reagent obtained in step (1) at a concentration of 2 μg / mL to the 96-well electrochemiluminescence microplate and incubate at 25°C with shaking for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0063] Step (4): Biotin-tyrosamide is deposited in situ based on TSA signal amplification technology.

[0064] Add a mixture of biotin-tyrosamide and H2O2 to the double-antibody sandwich system obtained in step (3). React for different times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min) under the action of catalyst HRP. After TSA signal amplification, a large amount of biotin is deposited in situ on the surface of HRP or its adjacent proteins, thereby increasing the amount of subsequent ruthenium-labeled streptavidin.

[0065] Step (5): Ruthenium-labeled streptavidin binds to biotin.

[0066] Add 100.0 μL of ruthenium-labeled streptavidin to the electrochemiluminescence microplate after the fourth step of treatment, and incubate with shaking at 25 °C for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0067] Step (6): Use an electrochemiluminescence immunoassay analyzer to quantitatively detect smegglutinin.

[0068] Tri-n-propylamine read buffer (100.0 μL / well) was added to the electrochemiluminescence microplate as a co-reactant, and the electrochemiluminescence values ​​were read at a wavelength of 620 nm. The data are as follows: Figure 4 As shown, a strong optical signal appears when the TSA signal amplification and processing time is 5 minutes; a strong optical signal also appears when the TSA signal amplification and processing time is 10 minutes. The intensity of the corresponding optical signal is not significantly different when the TSA signal amplification and processing time is 15 minutes, 20 minutes, 25 minutes, 30 minutes, and 40 minutes. Therefore, 10 minutes was chosen as the optimal time for the TSA response.

[0069] Example 4

[0070] To verify the analytical and detection performance of this method, a stable and reliable standard curve was established.

[0071] Step (1): Prepare HRP-labeled detection antibody.

[0072] Using CRIVVIN ® The HRP labeling kit was used to label semaglutide antibody, and the experimental procedure was strictly followed according to the manufacturer's instructions. The specific procedure is as follows: Using 0.01 M CB buffer, centrifuged at 1000×g for 8 min at 4 ℃. The ultrafiltration tube was equilibrated, and the mother solution of the antibody to be labeled was replaced sequentially. The antibody to be labeled was diluted to 2.0 mg / mL for later use. The antibody to be labeled: pre-activated HRP: labeling starter solution was mixed at a ratio of 250:100:63 (v / v / v) and incubated at 37 ℃ in the dark with shaking (500 rpm) for 2 h to conjugate the antibody with HRP. Then, 50.0 μL of reaction stop solution was added to the above mixture, and the mixture was incubated at 25 ℃ in the dark for 1 h to terminate the conjugation reaction. The HRP-antibody conjugate solution was purified by centrifugation with PBS buffer. Finally, an equal volume of labeling preservation solution was added, mixed well, and the HRP-labeled antibody was stored at 4 ℃.

[0073] Step (2): Prepare ruthenium-labeled streptavidin.

[0074] Using CRIVVIN ® A ruthenium-labeled kit was used to label streptavidin. First, a Ru activation solution was prepared by diluting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide sodium salt (NHS) to 100.0 mg / mL using 2-morpholine ethanesulfonate buffer (MES); simultaneously, lyophilized Ru powder was prepared to a concentration of 20.0 mg / mL. Then, a Ru activation solution was prepared at a ratio of Ru: NHS: EDC = 50:20:20 (V / V / V), and incubated at 25°C with shaking in the dark for 25 min to activate Ru. The streptavidin to be labeled was then mixed with the Ru activation solution at a 1:1 (mol / mol) ratio and incubated at 25°C with shaking in the dark for 2 h. After labeling, the mixture was purified by ultrafiltration.

[0075] Step (3): Construct a double-antibody sandwich immune system by introducing the TSA catalyst HRP.

[0076] Add 10 μg / mL and 100.0 μL of semaglutide capture antibody coating solution to a 96-well electrochemiluminescence microplate, and incubate overnight at 4°C. Add 300 μL of PBST working solution to each well, pat dry, and wash 3 times. Then, add 100.0 μL of semaglutide samples of different concentrations, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. Then, add the reagent obtained in step (1), 2 μg / mL and 100.0 μL of HRP-labeled detection antibody to the electrochemiluminescence microplate, and incubate at 25°C with shaking for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0077] Step (4): Biotin-tyrosamide is deposited in situ based on TSA signal amplification technology.

[0078] Add a mixture of biotin-tyrosamide and H2O2 to the double-antibody sandwich system obtained in step (3), and react for 10 min under the action of catalyst HRP. Through TSA signal amplification technology, a large amount of biotin is deposited in situ onto the surface of HRP or its adjacent proteins, thereby increasing the amount of subsequent ruthenium-labeled streptavidin.

[0079] Step (5): Ruthenium-labeled streptavidin binds to biotin.

[0080] Add 100.0 μL of ruthenium-labeled streptavidin to the electrochemiluminescence microplate obtained in step (4), and incubate with shaking at 25 °C for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0081] Step (6): Use an electrochemiluminescence immunoassay analyzer to quantitatively detect smegglutinin.

[0082] Tri-n-propylamine read buffer (100.0 μL / well) was added to the electrochemiluminescence microplate as a co-reactant, and the electrochemiluminescence values ​​were read at a wavelength of 620 nm. The data are as follows: Figure 5 As shown, the standard curve exhibits good linearity within the range of 20.0 to 4,000.0 pg / mL, with a detection limit of 20 pg / mL.

[0083] Example 5

[0084] Specificity verification, the specific process is as follows: Step (1): Prepare HRP-labeled detection antibody.

[0085] Using CRIVVIN ®The HRP labeling kit was used to label semaglutide antibody, and the experimental procedure was strictly followed according to the instructions. The specific procedure is as follows: using 0.01 M CB buffer, centrifuged at 4 ℃ at 1000×g for 8 min, the ultrafiltration tube was equilibrated and the stock solution of the antibody to be labeled was replaced sequentially, and the antibody to be labeled (semaglutide antibody) was diluted to 2.0 mg / mL for later use.

[0086] The antibody to be labeled: pre-activated HRP: labeling starter solution was mixed at a ratio of 250:100:63 (v / v / v) and incubated at 37 °C with shaking (500 rpm) in the dark for 2 h to conjugate the antibody with HRP. Then, 50.0 μL of reaction stop solution was added to the above mixture, and the mixture was incubated at 25 °C in the dark for 1 h to terminate the conjugation reaction. The HRP-antibody conjugate solution was purified by centrifugation using PBS buffer.

[0087] Finally, add an equal volume of labeling preservation solution, mix well, and store the HRP-labeled detection antibody at 4°C.

[0088] Step (2): Prepare ruthenium-labeled streptavidin.

[0089] Using CRIVVIN ® A ruthenium-labeled kit was used to label streptavidin. First, a Ru activation solution was prepared by diluting 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sodium N-hydroxythiosuccinimide (NHS) to 100.0 mg / mL using 2-morpholine ethanesulfonate buffer (MES); simultaneously, lyophilized Ru powder was prepared to a concentration of 20.0 mg / mL. Then, a Ru activation solution was prepared at a ratio of Ru:NHS:EDC = 50:20:20 (V / V / V), and incubated at 25°C with shaking in the dark for 25 min to activate Ru. The streptavidin to be labeled was then mixed with the Ru activation solution at a 1:1 (mol / mol) ratio and incubated at 25°C with shaking in the dark for 2 h. After labeling, the mixture was purified by ultrafiltration.

[0090] Step (3): Construct a double-antibody sandwich immune system by introducing the TSA catalyst HRP.

[0091] Add 10 μg / mL and 100.0 μL of semaglutide capture antibody coating solution to a 96-well electrochemiluminescence microplate, incubate overnight at 4°C, add 300 μL of PBST working solution to each well, pat dry, and wash 3 times; then add 12.5 ng / mL, 25 ng / mL, 50 ng / mL and 100 ng / mL of GLP-1 to the blank matrix, respectively, to prepare semaglutide to 3 ng / mL and 0.06 ng / mL, respectively. Add 100.0 μL of different concentrations of smegglutinin samples to an electrochemiluminescence plate and incubate at 25 °C with shaking for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times. Then add the reagent obtained in step (1), 2 μg / mL and 100.0 μL of HRP-labeled detection antibody to the electrochemiluminescence microplate and incubate at 25 °C with shaking for 1 h. After incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0092] Step (4): Biotin-tyrosamide is deposited in situ based on TSA signal amplification technology.

[0093] Add a mixture of biotin-tyrosamide and H2O2 to the double-antibody sandwich system obtained in step (3), and react for 10 min under the action of catalyst HRP. Through TSA signal amplification technology, a large amount of biotin is deposited in situ onto the surface of HRP or its adjacent proteins, thereby increasing the amount of subsequent ruthenium-labeled streptavidin.

[0094] Step (5): Ruthenium-labeled streptavidin binds to biotin.

[0095] Add 100.0 μL of ruthenium-labeled streptavidin to the electrochemiluminescence microplate obtained in step (4), and incubate with shaking at 25 °C for 1 h. After the incubation, wash the plate with PBST working solution, 300 μL / well, 3 times.

[0096] Step (6): Use an electrochemiluminescence immunoassay analyzer to quantitatively detect smegglutinin.

[0097] Tri-n-propylamine read buffer (100.0 μL / well) was added to the electrochemiluminescence microplate as a co-reactant, and the electrochemiluminescence values ​​were read at a wavelength of 620 nm. The data are as follows: Figure 5 As shown, GLP-1 was added to the blank matrix at concentrations of 12.5 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL, respectively, to investigate the accuracy of semaglutide at concentrations of 3 ng / mL and 0.06 ng / mL. At least 80% of the samples had %CV ≤ 20.0% and |%RE| ≤ 20.0%, indicating that the method has good specificity.

Claims

1. A method for detecting semaglutide using electrochemiluminescence immunoassay based on TSA signal amplification technology, characterized in that, Includes the following steps: Step 1: Preparation of HRP-labeled detection antibodies: HRP-labeled semaglutide antibody was used to obtain an HRP-labeled detection antibody reagent. The second step is the preparation of ruthenium-labeled streptavidin: Ruthenium labeling of streptavidin yielded ruthenium-labeled streptavidin. The third step involves adding the smegglutinin capture antibody coating solution to the electrochemiluminescence microplate and incubating it; then adding washing buffer to the electrochemiluminescence microplate for washing; adding the smegglutinin sample, incubating, and washing the plate; next, adding the HRP-labeled detection antibody reagent obtained in the first step to the electrochemiluminescence microplate, incubating, and then washing. Step 4: In-situ deposition of biotin-tyrosamide based on TSA signal amplification technology: A mixed solution of biotin-tyrosamide and H2O2 was added to an electrochemiluminescence microplate, and the signal was amplified by TSA. Step 5: Ruthenium-labeled streptavidin binds to biotin: Add ruthenium-labeled streptavidin to the electrochemiluminescence microplate, incubate, and wash the plate; Step 6: Quantitative detection of smegglutinin was performed using an electrochemiluminescence immunoassay analyzer. Add the electrochemiluminescence substrate solution to the electrochemiluminescence microplate and read the electrochemiluminescence value.

2. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 1, characterized in that, The concentration of the smegglutinin capture antibody coating solution is 1–10 μg / mL, and the concentration of the HRP-labeled detection antibody reagent is 0.5–4 μg / mL.

3. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 2, characterized in that, The concentration of the smegglutinin capture antibody coating solution is 10 μg / mL, and the concentration of the HRP-labeled detection antibody reagent is 2 μg / mL.

4. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 1, characterized in that, The first step involves labeling the smegglutinin antibody using an HRP labeling kit.

5. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 1, characterized in that, The second step involves labeling streptavidin using a ruthenium labeling kit.

6. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 1, characterized in that, The electrochemiluminescence substrate solution is tri-n-propylamine, a co-reactant.

7. The electrochemiluminescence immunoassay method for semaglutide based on TSA signal amplification technology according to claim 1, characterized in that, The fourth step, the TSA signal amplification process, takes 5 to 10 minutes.

8. A reagent kit, characterized in that, The reagents include HRP-labeled semaglutide detection antibody reagent, ruthenium-labeled streptavidin, semaglutide capture antibody coating solution, and a biotin-tyrosamide and H2O2 mixed solution.

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