A semaglutide antibody and an enzyme-linked immunoassay kit thereof

Abstract

The present application relates to the technical field of immune detection, and provides a semaglutide antibody, which is an anti-semaglutide monoclonal antibody and a single-domain nanobody VHH lacking light chains, and the amino acid sequence is shown as SEQ ID NO. 1. The present application also provides application of the above antibody in preparation of a semaglutide enzyme-linked immune detection kit, and simultaneously provides a semaglutide enzyme-linked immune detection kit. The antibody of the present application has high affinity, high specificity and good stability, can accurately recognize semaglutide and avoid cross-reaction with endogenous GLP-1, other GLP-1 receptor agonists and the like analogs, and is suitable for detection scenes in complex biological matrices; the corresponding enzyme-linked immune kit is simple in operation, sensitive and accurate in detection, and can effectively solve the problems of insufficient antibody specificity, complex detection process, high cost and difficult to be applied in scale of the existing semaglutide detection technology.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and more particularly to a semaglutide antibody and its enzyme-linked immunosorbent assay kit. Background Technology

[0002] In recent years, the global incidence of diabetes, especially type 2 diabetes mellitus (T2DM), has continued to rise, becoming a chronic metabolic disease that seriously threatens human health. Its core pathological features include insulin resistance and progressive decline in pancreatic β-cell function, ultimately leading to a persistent hyperglycemic state. According to data released by the International Diabetes Federation (IDF), the number of people with diabetes worldwide has exceeded 500 million, and the disease is showing a trend towards affecting younger people. Long-term uncontrolled hyperglycemia can trigger a series of serious complications such as cardiovascular and cerebrovascular diseases, kidney damage, retinopathy, and neurological damage, not only severely reducing patients' quality of life but also imposing a heavy economic burden on global healthcare systems. Therefore, developing safe and effective treatments and establishing corresponding precise monitoring and detection technologies are of great significance for the clinical management of T2DM.

[0003] Glucagon-like peptide-1 (GLP-1) receptor agonists are a novel class of hypoglycemic drugs with unique mechanisms. They improve glucose metabolism through multiple pathways, including enhancing glucose-dependent insulin secretion, inhibiting glucagon release, delaying gastric emptying, and regulating appetite, and have become one of the core drug classes for the treatment of type 2 diabetes mellitus (T2DM). Semaglutide, a representative drug in this class, is a synthetic analog obtained by modifying the amino acid and fatty acid chains of natural GLP-1. Compared with the earlier marketed liraglutide, semaglutide has a longer in vivo half-life, better hypoglycemic and weight-loss effects, and can be administered once a week, significantly improving patient adherence. Currently, semaglutide has been approved for the treatment of T2DM and also shows broad application prospects in areas such as obesity intervention and reduction of cardiovascular disease risk, with clinical demand continuing to grow.

[0004] However, with the increasingly widespread clinical application of semaglutide, the clinical need for monitoring its blood drug concentration and detecting its immunogenicity is becoming increasingly urgent. On the one hand, as a large-molecule peptide drug, semaglutide is affected by various factors such as individual metabolic differences, concomitant medications, and liver and kidney function, resulting in significant differences in pharmacokinetic characteristics among different patients. Too low a serum drug concentration can lead to poor hypoglycemic effect and ineffective blood glucose control, while too high a concentration may increase the risk of adverse reactions such as gastrointestinal discomfort and pancreatitis. Therefore, developing rapid, sensitive, and highly specific detection methods to provide data support for adjusting individualized clinical medication regimens is crucial for ensuring treatment efficacy and medication safety. On the other hand, long-term exposure to peptide drugs in vivo may induce the production of anti-drug antibodies (ADAs). These antibodies may weaken drug efficacy and even trigger immune-related adverse events. Therefore, long-term monitoring of the immunogenicity of semaglutide has also become an important aspect of safe clinical medication use.

[0005] Currently, detection techniques for semaglutide are mainly divided into two categories: liquid chromatography-mass spectrometry (LC-MS / MS) and immunological detection methods. LC-MS / MS, with its high qualitative and quantitative accuracy and high sensitivity, is often used as a reference detection method. However, this technique suffers from drawbacks such as cumbersome sample pretreatment procedures, high equipment costs, high operational expertise requirements, and low throughput, making it difficult to meet the large-scale testing needs of routine clinical laboratories. In contrast, immunological detection methods based on antigen-antibody specific binding, especially enzyme-linked immunosorbent assays (ELISA), offer significant advantages such as ease of operation, high throughput, controllable cost, and intuitive results, making them more suitable for routine clinical monitoring and pharmacokinetic studies in drug development. However, developing highly specific antibodies against semaglutide and constructing stable and reliable ELISA assay kits based on these antibodies remains a significant challenge. Firstly, semaglutide has a relatively small molecular weight (approximately 4 kDa) and high sequence homology with endogenous human GLP-1, resulting in relatively weak antigenicity and difficulty in inducing the body to produce high-affinity, high-specificity antibodies, greatly complicating antibody screening and preparation. Secondly, to ensure detection accuracy, the antibody must be able to strictly distinguish semaglutide from natural GLP-1 and other structurally similar GLP-1 receptor agonists (such as dulaglutide and liraglutide) to avoid cross-reactions leading to biased test results. Thirdly, clinical test samples are often complex biological matrices such as serum and plasma; antibodies must maintain good stability and detection sensitivity in these matrices to accurately capture the target drug molecule and meet the practical needs of clinical sample testing. Therefore, developing high-quality semaglutide-specific antibodies and constructing corresponding ELISA assay kits is of significant technical value for promoting the rational clinical application, efficacy evaluation, and pharmacokinetic and pharmacodynamic studies of this drug.

[0006] Currently, publicly reported methods for detecting semaglutide primarily focus on chromatography or mass spectrometry, with relatively little research on highly specific immunoantibodies and their corresponding enzyme-linked immunosorbent assay (ELISA) kits. Although some commercial institutions have launched universal detection reagents for GLP-1 or its analogues, these reagents generally suffer from insufficient specificity (easily cross-reacting with other GLP-1 analogues), limited sensitivity (difficult to detect low-concentration samples), and narrow detection range, failing to meet the clinical and research needs for accurate semaglutide detection. Therefore, developing an antibody capable of highly specifically recognizing semaglutide and establishing a stable, sensitive, and convenient ELISA kit based on this antibody would not only effectively address the shortcomings of existing technologies but also provide strong technical support for clinical drug monitoring, efficacy evaluation, immunogenicity analysis, and related drug development of semaglutide. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a semaglutide antibody and its enzyme-linked immunosorbent assay (ELISA) kit. The antibody has high affinity, high specificity and good stability, and can accurately identify semaglutide and avoid cross-reaction with endogenous GLP-1, other GLP-1 receptor agonists and other analogues. At the same time, it is suitable for detection scenarios in complex biological matrices. The corresponding ELISA kit is easy to operate and has high sensitivity and accuracy, which can effectively solve the problems of insufficient antibody specificity, complex detection process, high cost and difficulty in large-scale application of existing semaglutide detection technology.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A semaglutide antibody, wherein the antibody is an anti-semaglutide monoclonal antibody and is a single-domain nanobody (VHH) lacking a light chain, and the amino acid sequence is shown in SEQ ID NO.1.

[0009] As one of the preferred embodiments of the present invention, the antibody exists in the form of a single VHH, an N-terminal fusion expression of the antibody Fc segment, or a C-terminal fusion expression of the antibody Fc segment.

[0010] As one of the preferred embodiments of the present invention, the Fc segment is the Fc segment of a human, mouse, sheep or rabbit IgG subtype antibody.

[0011] The application of the above-mentioned semaglutide antibody in the preparation of a semaglutide enzyme-linked immunosorbent assay kit.

[0012] As one of the preferred embodiments of the present invention, the semaglutide antibody is used as the coating antibody, and BSA-semaglutide-labeled horseradish peroxidase, OVA-semaglutide-labeled horseradish peroxidase or KLH-semaglutide-labeled horseradish peroxidase are used as labeling proteins in the enzyme-linked immunosorbent assay kit.

[0013] A semaglutide enzyme-linked immunosorbent assay kit contains the semaglutide antibody described above, wherein the semaglutide antibody serves as a coating antibody.

[0014] As one of the preferred embodiments of the present invention, it further includes a labeled protein, a semaglutide standard, a concentrated washing solution, a TMB colorimetric solution, and a stop solution; the labeled protein is BSA-semaglutide-labeled horseradish peroxidase, OVA-semaglutide-labeled horseradish peroxidase, or KLH-semaglutide-labeled horseradish peroxidase.

[0015] As one of the preferred embodiments of the present invention, the concentration range of the semaglutide standard is 0.2~100 ng / mL, and PBS buffer containing 1% BSA is used as the diluent. The concentrated washing solution is a PBS buffer containing 0.1% Tween-20 at pH 7.4, which should be diluted 10 times with deionized water before use. The terminating solution is 1M H2SO4.

[0016] As one of the preferred embodiments of the present invention, a blocking solution is also included; the blocking solution is a PBS buffer containing 1% BSA, 5% sucrose and 0.05% Proclin-300, with a pH of 7.4.

[0017] As one of the preferred embodiments of the present invention, the detection limit of the kit is 0.16 ng / mL, the coefficient of variation of intra-batch precision is less than 8%, and the spiked recovery rate is 97.3%~105.0%.

[0018] The advantages of this invention compared to the prior art are: (1) The antibody structure of the present invention is special and has excellent stability. It is a single-domain nanobody (VHH) lacking light chain and has a molecular weight of only about 15 kDa, which is much smaller than that of traditional antibodies. When used as a coating antibody in an enzyme-linked immunosorbent assay (ELISA) platform, it is not easily affected by environmental factors such as temperature and pH, and can still maintain good structural stability in complex detection systems.

[0019] (2) The antibody of this invention has outstanding affinity and specificity. Its unique amino acid sequence (SEQ ID NO.1) ensures high affinity binding to smegglutinin molecules, with an EC50 value of 6.675 ng / mL. It can also strictly distinguish smegglutinin from structural analogs such as endogenous GLP-1, glucagon, insulin, and exenatide, with no cross-reactivity, laying the foundation for high-precision and high-reliability immunoassay.

[0020] (3) The antibody of the present invention has a flexible and adaptable form. It can exist alone in the form of VHH, or it can be fused with the Fc segment of human, mouse, sheep or rabbit IgG subtype antibody at the N-terminus or C-terminus. After fusion, it still maintains excellent binding activity and can be adapted to different needs such as routine detection and high-sensitivity detection, as well as various experimental systems such as human sample detection and animal experimental research, thus expanding the application scenarios of the antibody.

[0021] (4) The kit of the present invention is practical, efficient and easy to industrialize. The enzyme-linked immunosorbent assay kit constructed based on the antibody (with the nanobody as the coating antibody and BSA-semaglutide-HRP as the label) adopts a "one-step incubation" process, which is simple to operate, has high detection throughput and does not require complicated sample pretreatment. The detection range covers 0.2~100ng / mL, the detection limit is as low as 0.16ng / mL, the intra-assay precision coefficient of variation (CV%) is less than 8%, the spiked recovery rate is between 97.3% and 105.0%, and the detection results are accurate, reliable and reproducible. It provides a new solution for the quantitative detection of semaglutide that is efficient, stable and easy to industrialize, and solves the problems of cumbersome steps, weak signal or poor stability that may exist in existing kits. At the same time, it provides strong technical support for the clinical drug monitoring, efficacy evaluation, immunogenicity analysis and related drug development of semaglutide. Attached Figure Description

[0022] Figure 1 This is an SDS-PAGE image of the VHH form of the semaglutide monoclonal antibody of this invention (in the image, lane "M" is a 10~180kDa protein marker; lane "1" is the anti-semaglutide VHH monomer). Figure 2 This is an ELISA affinity assay diagram of the anti-semaglutide monoclonal antibody and semaglutide of the present invention. Figure 3 This is an SDS-PAGE image of the semaglutide monoclonal antibody VHH-Fc of this invention (in the image, lane "M" is a 25~180kDa protein marker; lane "1" is the anti-semaglutide VHH-Fc fusion antibody). Figure 4 This is a comparison chart of detection results for Fc fusion antibodies from different sources; Figure 5 This is the calibration curve diagram of the present invention; Figure 6 This is a linear correlation graph of the enzyme-linked immunosorbent assay kit of the present invention. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.

[0024] Example 1: Screening of anti-semaglutide nanobody (VHH): 1. Antigen coating 200 pmol of BSA-semaglutide-biotin (prepared by biotin-NHS ester labeling after conjugation of semaglutide with BSA) was incubated with 20 μg of M280-streptavidin magnetic beads at room temperature for 1 h to allow the antigen to bind to the surface of the magnetic beads. After washing three times with PBS buffer, 5% skim milk powder-PBS buffer (PBST) was added for blocking for 1 h to reduce non-specific binding.

[0025] 2. Phage library binding The pre-constructed synthetic nanobody phage display library was diluted to 10. 12 Add pfu / mL to the above magnetic beads and incubate with gentle shaking at room temperature for 1 hour to ensure that the phage and antigen are fully bound.

[0026] 3. Washing and elution Discard unbound phages and wash 10 times with PBST containing 0.05% Tween-20 to remove non-specifically bound phages. Add 0.1M glycine-HCl elution buffer (pH 2.2), incubate at room temperature for 10 min, and immediately neutralize the elution buffer with 1M Tris-HCl (pH 9.0) to collect antigen-bound phages.

[0027] 4. Amplification and Enrichment The eluted phages were used to infect logarithmically growing E. coli TG1 competent cells and cultured at 37°C in a shaker for 30 min. Amplification was then performed using 2×YT medium containing ampicillin and glucose. The phage supernatant was concentrated using PEG / NaCl precipitation. This coating, binding, elution, and amplification process was repeated 2-3 times, increasing the number of washes and the Tween-20 concentration in each round to enhance the selection pressure and thus enrich a population of high-affinity smegglutinin-binding phages.

[0028] 5. Monoclonal screening and identification Enriched bacteriophages were used to infect *E. coli*, and single-clone colonies were selected to induce the expression of nanobody fragments. Identification was performed using a soluble supernatant ELISA method: BSA-coated wells were used as detection wells, and PBS-coated wells were used as control wells. The binding activity and specificity of different clones to semaglutide were detected. Finally, OD... 450 Positive clones with significantly elevated levels and no obvious signal in the control wells.

[0029] 6. Sequencing analysis The nanobody genes of positive clones were sequenced to analyze the diversity of CDR region sequences, and representative clones were selected for subsequent expression and purification.

[0030] This embodiment ultimately yielded an antisemaglutide nanobody (VHH) with the amino acid sequence shown in SEQ ID NO.1. This antibody has the characteristics of small molecular weight, high stability, and a single antigen binding site, making it suitable for the application requirements of subsequent enzyme-linked immunosorbent assay (ELISA) platforms.

[0031] Example 2: Gene synthesis and vector construction of anti-semaglutide nanobody (VHH): 1. Based on the amino acid sequence shown in SEQ ID NO.1, and according to the codon preference of E. coli, the DNA sequence encoding the nanobody was chemically synthesized (shown in SEQ ID NO.2).

[0032] 2. Using restriction endonucleases Nco I and Xho I. The synthesized DNA fragment and expression vector pET-22b (+) were double-digested. After recovering the digestion products, they were ligated with T4 DNA ligase and transformed into E. coli DH5α competent cells to construct the recombinant plasmid pET-22b-VHH.

[0033] 3. The recombinant plasmid was sequenced and verified. The plasmid with the correct sequence was transformed into Escherichia coli BL21 (DE3) competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C to obtain positive expression strains.

[0034] Example 3: Expression and purification of anti-smegglutinin nanobody (VHH): 1. Select a single colony of the positive expression strain obtained in Example 2 and inoculate it into LB liquid medium containing 100 μg / mL ampicillin. Incubate at 37°C with shaking at 220 rpm until OD reaches 100%. 600 The value reaches 0.6~0.8.

[0035] 2. Add isopropyl-β-D-thiogalactoside (IPTG) to the culture medium to a final concentration of 0.5 mM and induce expression at 28°C for 16 h.

[0036] 3. After the induction expression is completed, the bacterial cells are collected by centrifugation, and after sonication, the supernatant is collected by centrifugation.

[0037] 4. The histidine-tag fusion nanobody in the supernatant was purified using a Ni-NTA affinity chromatography column and eluted with an elution buffer containing 20 mM Tris, 150 mM NaCl, 5% glycerol and 500 mM imidazole.

[0038] 5. The purity of the eluted fraction was determined by SDS-PAGE electrophoresis, and the results are as follows: Figure 1As shown, a single target band appeared at approximately 15 kDa (the band had a reasonable offset of ±2 kDa due to errors in the His-tag label and electrophoresis system), with no obvious extraneous bands, indicating that high-purity anti-semaglutide nanobody (VHH) was successfully obtained.

[0039] Example 4: Verification of direct binding between anti-semaglutide nanobody and semaglutide (ELISA method): 1. Dilute semaglutide to 2 μg / mL with PBS buffer, and coat 100 μL per well in a 96-well microplate. Incubate overnight at 4°C.

[0040] 2. After washing the plate three times, add PBS solution containing 3% BSA and block at 37°C for 2 hours.

[0041] 3. Wash the plate and add a series of concentration gradients (0.1 nM to 100 nM) of the purified antisemaglutide nanobody (VHH) from Example 3, and incubate at 37°C for 1 h.

[0042] 4. Wash the plate, add mouse anti-His tag primary antibody, and incubate at 37°C for 1 hour.

[0043] 5. Wash the plate, add HRP-labeled goat anti-mouse secondary antibody, and incubate at 37°C for 1 hour.

[0044] 6. Wash the plate, add TMB substrate solution for color development, terminate the reaction with 2M H2SO4, and measure the absorbance value at a wavelength of 450nm.

[0045] See results Figure 2 The nanobody can bind specifically to smegglutinin in a dose-dependent manner, and its EC50 value is calculated to be 6.675 ng / ml, indicating high affinity.

[0046] Example 5: Construction and expression of anti-semaglutide-Fc fusion antibody (VHH-Fc): 1. The DNA sequence encoding VHH shown in SEQ ID NO.1 (SEQ ID NO.2) was linked to the DNA sequence of the human IgG1 Fc segment (a known sequence) via the flexible linker peptide (G4S)3. The fusion gene was chemically synthesized in the order of “VHH-(G4S)3-Fc”. At the same time, restriction enzyme sites (Hind III and Xba I) matching the mammalian expression vector pcDNA3.4 were introduced at both ends of the gene.

[0047] 2. The synthesized fusion gene and the commercially available mammalian expression vector pcDNA3.4 were double-digested with Hind III and Xba I enzymes; the target fragment and vector backbone were recovered by electrophoresis and ligated overnight at 16°C using T4 DNA ligase; the cells were transformed into E. coli DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated overnight at 37°C; single colonies were picked, the recombinant plasmid was extracted and sequenced for verification, and the recombinant expression plasmid pcDNA3.4-VHH-Fc was obtained.

[0048] 3. Using polyethyleneimine (PEI) transfection reagent, the recombinant plasmid pcDNA3.4-VHH-Fc was transfected into Expi293F mammalian suspension cells. The cells were cultured at 37°C and 8% CO2 for 7 days, and the cell culture supernatant was collected.

[0049] 4. The Fc fusion protein in the supernatant was captured and purified using a Protein A affinity column. A Protein A affinity column pre-equilibrated with Binding Buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) was used. After loading and filtering the sample, the supernatant was washed with 5 column volumes of Binding Buffer to remove impurities, followed by elution with Elution Buffer (0.1 M citric acid, pH 3.0). The eluent was collected and immediately neutralized to pH 7.0 with 1 M Tris-HCl (pH 9.0).

[0050] 5. The purified product was identified by SDS-PAGE electrophoresis, and the results are as follows: Figure 3 As shown, a dimeric form of antisemaglutide-Fc fusion antibody (VHH-Fc) with a molecular weight of approximately 70 kDa was obtained.

[0051] Example 6: Preparation and performance comparison of VHH-Fc fusion antibodies from different species: 1. Following the method in Example 5, the human IgG1 Fc fragment was replaced with mouse IgG1 Fc, rabbit IgG Fc, and sheep IgG1 Fc fragments (all known sequences), respectively. Corresponding recombinant expression plasmids were constructed, transfected into Expi293F cells, and purified to obtain anti-semaglutide antibodies in four different species Fc fusion forms.

[0052] 2. Different purified Fc fusion antibodies were used as capture antibodies and coated with microplates according to the method in Example 4.

[0053] 3. Using the same HRP markers and detection procedures, compare their detection signals and signal-to-noise ratios.

[0054] See results Figure 4All Fc fusion forms of antibodies can effectively bind to semaglutide and generate a stable detection signal.

[0055] Example 7: Assembly of the Smegglutide ELISA kit (using VHH as the capture antibody): 1. Preparation of coating plate The anti-semaglutide nanobody (VHH, purity >99%) purified in Example 3 was diluted to 5 μg / mL with coating buffer (0.05 M carbonate buffer, pH 9.6) and coated into white microplates for ELISA, 100 μL per well, and incubated overnight at 4°C. After washing, 250 μL of PBS buffer containing 5% sucrose and 1% BSA was added to each well for blocking, and the plates were dried at 37°C for 2 h.

[0056] 2. Labeling preparation The BSA-smegglutinin-SA-HRP complex was prepared by coupling polymerization of BSA-smegglutinin-HRP using the streptavidin-biotin method, and the free components were removed by purification.

[0057] 3. Reagent preparation Prepare the working concentration of ABEI-semaglutide using the kit dilution solution (PBS buffer containing 1% BSA).

[0058] 4. Reagent kit assembly The coated plate, HRP marker, Smegglutinin series standards (0.2~100.0 ng / mL), concentrated wash buffer (PBS buffer containing 0.1% Tween-20, pH 7.4, diluted 10 times with deionized water before use), TMB chromogenic solution, 1M H2SO4 stop solution, and blocking buffer (PBS buffer containing 1% BSA, 5% sucrose and 0.05% Proclin-300, pH 7.4) were assembled into an enzyme-linked immunosorbent assay kit.

[0059] Example 8: Establishment of the standard curve and performance testing of the enzyme-linked immunosorbent assay kit: 1. Dilute the smegglutide standard with negative serum to a series of concentrations (0~100ng / mL).

[0060] 2. Add 50 μL of standard and 50 μL of HRP label to the wells of the reagent plate and incubate at 37°C with shaking for 30 minutes.

[0061] 3. Discard the liquid inside the well, wash the plate 5 times with detergent, and pat it dry.

[0062] 4. Inject 50 μL of TMB into each well sequentially, incubate for 15 min, and then add 50 μL of stop solution to each well.

[0063] 5. Immediately measure the absorbance (OD) of each well at a wavelength of 450 nm using an ELISA reader. 450 ), with the logarithm of the standard concentration as the x-axis, OD 450 The value is the ordinate (Y). A standard curve for the four-parameter logistic (4-PL) fitting is plotted. The results are shown in [Figure / Reference]. Figure 5 The fitted standard curve has a linear range covering 0.2–100 ng / mL, a correlation coefficient (R²) greater than 0.99, and a good dose-response relationship, meeting the requirements for quantitative detection.

[0064] 6. Sensitivity testing: Determine the OD of 20 zero-concentration standards. 450 The values ​​were calculated, and the mean (Mean) and standard deviation (SD) were calculated. The limit of detection (LoD) was calculated using the formula Mean + 2.5 × SD. Combined with the standard curve, the limit of detection of the kit was found to be 0.16 ng / mL, indicating that the kit has high detection sensitivity.

[0065] Example 9: Validation of reagent kit precision and accuracy: 1. Intra-batch precision verification Three quality control samples of smegglutinin at low, medium, and high concentrations were selected, with concentrations of 1.5 ng / mL, 15 ng / mL, and 80 ng / mL, respectively. Each concentration was tested repeatedly in 24 wells on the same ELISA plate, following the detection procedure in Example 8. The coefficient of variation (CV%) of the test values ​​for each concentration was calculated.

[0066] The results are shown in Table 1.

[0067] Table 1. Results of Coefficient of Variation

[0068] The results showed that the intra-batch CV% for low, medium, and high concentration samples were 6.34%, 4.84%, and 4.10%, respectively, all less than 8%, indicating that the kit has excellent intra-batch repeatability and the detection precision meets the industry standards for immunoassay reagents.

[0069] 2. Verification of testing accuracy Serum samples from negative healthy individuals were collected, and semaglutide standards of 1.0 ng / mL, 10.0 ng / mL, and 50.0 ng / mL were added to prepare three spiked samples. Each spiked sample was tested three times. The actual detection value was determined according to the procedure in Example 8, and the spiked recovery rate was calculated. The recovery rate formula is: Recovery rate (%) = (Actual detection value - Negative serum background value) / Theoretical spiked value × 100%.

[0070] The results are shown in Table 2.

[0071] Table 2. Spike Recovery Results

[0072] The results showed that the recovery rates of the three spiked samples were 97.3%–105.0%, with an average recovery rate of 102.0%, indicating that the kit has high detection accuracy, the serum matrix has no significant interference with the detection results, and it can accurately reflect the concentration of semaglutide in the actual samples.

[0073] 3. Verification of linear correlation Linear regression analysis was performed on the measured concentrations and theoretical concentrations of the spiked and quality control samples. A linear correlation plot was created with the measured concentration as the x-axis (X) and the theoretical concentration as the y-axis (Y) (see Figure 6). The fitted linear equation was y = 1.0155x - 0.3814, with a correlation coefficient R0. 2 =0.9997. This indicates that the linear fit between the measured and theoretical values ​​is extremely high, further verifying the quantitative accuracy and reliability of the kit within the detection range of 0.2~100 ng / mL.

[0074] Example 10: Validation of reagent kit specificity: 1. Endogenous polypeptides and similar drugs with structures similar to semaglutide were selected as interferons, including GLP-1, glucagon, insulin, and exenatide. Each interferon was prepared into a high-concentration solution of 1000 ng / mL using the kit diluent. At the same time, a 0 ng / mL semaglutide negative control, and 5 ng / mL and 20 ng / mL semaglutide positive quality control controls were set up.

[0075] 2. Following the detection procedure in Example 8, each interfering solution, negative control, and positive control were tested. Each sample was tested three times, and the OD values ​​were recorded. 450 The values ​​were then calculated and the average detection concentration was determined.

[0076] The results are shown in Table 3.

[0077] Table 3. Specific Detection Results

[0078] The results showed that the average detection concentration of each interfering substance was lower than the detection limit of the kit (0.16 ng / mL), and there was no significant difference from the negative control. The detection value of the positive control was within ±5% of the theoretical value, indicating that the kit of the present invention has high specificity for smegglutinin and will not cross-react with structural analogs such as GLP-1 and glucagon, which can effectively avoid false positive results in clinical testing.

[0079] Example 11: Preparation of lyophilized reagents: Take the HRP-labeled working solution prepared in Example 7 and mix it evenly with a lyophilization protectant (such as trehalose or BSA); quantitatively dispense it into the wells of a microplate or into a special lyophilization bottle; place it in a freeze dryer for pre-freezing, primary drying, and desorption drying to produce lyophilized powder; when using, redissolve it with deionized water or a special reconstitution solution.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A semaglutide antibody, characterized in that, The antibody is an anti-semaglutide monoclonal antibody, and is a single-domain nanobody VHH lacking a light chain, with the amino acid sequence shown in SEQ ID NO.

1.

2. The semaglutide antibody according to claim 1, characterized in that, The antibody may exist in the form of a standalone VHH, an N-terminal fusion expression of the antibody Fc segment, or a C-terminal fusion expression of the antibody Fc segment.

3. The semaglutide antibody according to claim 2, characterized in that, The Fc segment is the Fc segment of human, mouse, sheep, or rabbit IgG subtype antibodies.

4. The use of the semaglutide antibody according to any one of claims 1 to 3 in the preparation of a semaglutide enzyme-linked immunosorbent assay kit.

5. The application according to claim 4, characterized in that, The semaglutide antibody is used as the coating antibody, and BSA-semaglutide-labeled horseradish peroxidase, OVA-semaglutide-labeled horseradish peroxidase, or KLH-semaglutide-labeled horseradish peroxidase are used as labeling proteins in the enzyme-linked immunosorbent assay kit.

6. A semaglutide enzyme-linked immunosorbent assay kit, characterized in that, The antibody comprises the semaglutide antibody according to any one of claims 1 to 3, wherein the semaglutide antibody is used as a coating antibody.

7. The smegglutide enzyme-linked immunosorbent assay kit according to claim 6, characterized in that, It also includes a labeled protein, semaglutide standard, concentrated washing solution, TMB colorimetric solution, and stop solution; the labeled protein is BSA-semaglutide-labeled horseradish peroxidase, OVA-semaglutide-labeled horseradish peroxidase, or KLH-semaglutide-labeled horseradish peroxidase.

8. The smegglutide enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, The concentration range of the smegglutinin standard is 0.2~100 ng / mL, and it is diluted with PBS buffer containing 1% BSA; the concentrated washing solution is PBS buffer containing 0.1% Tween-20 at pH 7.4, and it is diluted 10 times with deionized water before use; the stop solution is 1M H2SO4.

9. The smegglutide enzyme-linked immunosorbent assay kit according to claim 7, characterized in that, It also includes a blocking solution; the blocking solution is a PBS buffer containing 1% BSA, 5% sucrose and 0.05% Proclin-300, pH 7.

4.

10. The smegglutide enzyme-linked immunosorbent assay kit according to claim 6, characterized in that, The kit has a detection limit of 0.16 ng / mL, an intra-batch precision coefficient of variation of less than 8%, and a spiked recovery rate of 97.3%–105.0%.

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