A method for detecting the content of semaglutide based on electrochemiluminescence immunoassay, a kit for detecting semaglutide and a monoclonal antibody pair

Abstract

The present application relates to a kind of based on electrochemiluminescence immunoassay semeglu-tide content detection method, the kit for detecting semeglu-tide and monoclonal antibody pair, it solves the technical problem that the prior art through LC-MS / MS determination semeglu-tide content exists and is complex, detection flux is low, impurity residue, sample loss amount is big and traditional ELISA detection sensitivity is low, it is by the way that one monoclonal antibody reagent AS-MAB01 is coated on electrochemiluminescence microplate, specifically recognizes and combines semeglu-tide in matrix, then another monoclonal antibody AS-MAB02 labeled with ruthenium is added to form sandwich complex, finally, high sensitivity semeglu-tide quantitative detection purpose is achieved by electrochemiluminescence principle.The present application can be used for the quantitative detection of semeglu-tide in mouse, monkey and human-derived serum samples.

Description

Technical Field

[0001] This invention relates to the field of detection technology for blood concentration of polypeptide drugs, and more specifically, to a method for detecting semaglutide content based on electrochemiluminescence immunoassay, a kit for detecting semaglutide, and a monoclonal antibody pair. Background Technology

[0002] Semaglutide, developed by Novo Nordisk, is a novel long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA). It is a 31-amino acid polypeptide that shares 94% structural homology with natural GLP-1 to avoid immunogenicity. Based on natural GLP-1, semaglutide undergoes structural modifications that reduce renal clearance and prolong the degradation half-life of DPP-4 enzymes, while also enhancing its affinity for the GLP-1 receptor.

[0003] Smegglutinin, a GLP-1 receptor agonist, promotes insulin synthesis by stimulating GLP-1 receptors and exerts its hypoglycemic effect by promoting insulin secretion in a glucose concentration-dependent manner. It also inhibits glucagon secretion, regulating blood sugar. Furthermore, it suppresses appetite, delays gastric emptying, and increases satiety. Clinically, it has been approved for the treatment of type 2 diabetes and obesity.

[0004] Peptide drugs differ significantly from small-molecule drugs in their physicochemical properties. Due to their strong biological activity, they are typically administered in small doses, resulting in low in vivo exposure. Therefore, high sensitivity is required for detection methods. Furthermore, biological samples often contain numerous interfering components with similar physicochemical properties to the analyte, leading to significant endogenous interference with protein and peptide drugs, further necessitating high specificity in detection methods. Currently, the determination of semaglutide content primarily employs liquid chromatography-mass spectrometry (LC-MS / MS). While this technique offers high specificity and a wide linear range, it suffers from drawbacks such as complex sample pretreatment, low throughput, residual impurities, and significant sample loss. Additionally, commercially available ELISA kits also exhibit relatively low detection sensitivity. Summary of the Invention

[0005] This application aims to address the technical problems of existing technologies for determining semaglutide content using LC-MS / MS, such as complex pretreatment, low detection throughput, residual impurities, large sample loss, and low sensitivity of traditional ELISA. It provides a high-precision and accurate electrochemiluminescence immunoassay-based method for detecting semaglutide content, a kit for detecting semaglutide, and a monoclonal antibody pair.

[0006] The electrochemiluminescence immunoassay platform combines sensitive electrochemiluminescence detection technology with highly specific immunoassay technology. It features stable and reliable detection results, and its sensitivity, precision, and accuracy are superior to traditional ELISA. Compared with LC-MS / MS technology, its biggest advantages are high sensitivity, high-throughput sample detection, and simple operation.

[0007] This disclosure first screens nearly 20 monoclonal antibodies using mouse immunization technology, and then further pairs them into key reagents using monoclonal antibodies AS-MAB01 and AS-MAB02 as paired antibodies. AS-MAB01 is used as the coating antibody and AS-MAB02 is used as the detection antibody. A sensitive and simple method is established using an electrochemiluminescence immunoassay platform to detect the content of smegglutinin in serum, which can then be used for clinical / pre-pharmacokinetic studies of this drug.

[0008] A first aspect of this disclosure provides a monoclonal antibody pair comprising monoclonal antibody AS-MAB01 and monoclonal antibody AS-MAB02, wherein the light chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.1 and the heavy chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.5; the light chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.2 and the heavy chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.6.

[0009] A second aspect of this disclosure provides a kit for detecting semaglutide, comprising a monoclonal antibody pair, a washing buffer, a labeling reagent, an electrochemiluminescent microplate, and an electrochemiluminescent substrate solution;

[0010] The monoclonal antibody pair comprises monoclonal antibody AS-MAB01 and monoclonal antibody AS-MAB02. The light chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.5. The light chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.2, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.6.

[0011] Preferably, the labeling reagent is a ruthenium-labeled reagent.

[0012] Preferably, the washing buffer is Tween 20 phosphate buffer.

[0013] Preferably, the kit for detecting smegglutinin also includes a sample diluent.

[0014] More preferably, the sample diluent is a Casein buffer. Even more preferably, the Casein buffer contains 0.01% to 0.5% Tween 20; even more preferably, the Casein buffer contains 0.05% Tween.

[0015] Preferably, the electrochemiluminescence substrate solution is an electrochemiluminescence substrate solution containing tri-n-propylamine. More preferably, the tri-n-propylamine content is 1% to 10%; even more preferably, the tri-n-propylamine content is 5%.

[0016] A third aspect of this disclosure provides a method for detecting semaglutide content based on electrochemiluminescence immunoassay, comprising the following steps:

[0017] The first step is the preparation of monoclonal antibodies and the screening of antibody pairs;

[0018] Step 1: Mice were immunized with smegglutinin standard to screen for 4 monoclonal antibodies.

[0019] Step 2: Then, using the ELISA method, each monoclonal antibody and its corresponding HRP-labeled antibody are paired up. By comparing the S / N values, the pair with the largest S / N value is the optimal antibody pair, thus obtaining the optimal antibody pair: coating antibody and detection antibody.

[0020] The second step is to prepare ruthenium-labeled monoclonal detection antibody reagents;

[0021] The third step involves using an electrochemiluminescence immunoassay analyzer for detection.

[0022] Step 1: Coat the monoclonal antibody reagent in a microplate at a concentration of 2.0–10.0 ug / mL and incubate at 4°C for 16–24 hours.

[0023] Step 2: Wash three times with Tween 20 phosphate buffer, drain the residual liquid, and then block with 1% Casein at room temperature for 1-2 hours.

[0024] Step 3: Prepare the samples, quality control samples, and test samples for fitting the standard curve;

[0025] Use serum to prepare standard curve samples and quality control samples. Dilute the test samples with sample diluent, with a minimum dilution factor of 1 to 100, to obtain standard curve samples, quality control samples and test samples.

[0026] Step 4: Add the sample used for fitting the standard curve, the quality control sample, and the test sample to the microplate, 100 μL / well, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST.

[0027] Step 5: Add ruthenium-labeled monoclonal detection antibody reagent to the microplate, 100 μL / well, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST;

[0028] Step 6: Add 100 μL of tri-n-propylamine, a co-reactant, to the microplate and read the results using an electrochemiluminescence immunoassay analyzer.

[0029] Preferably, in the first step, the four monoclonal antibodies screened are: AS-MAB01, AS-MAB02, AS-MAB03, and AS-MAB04. The light chain amino acid sequence of AS-MAB01 is shown in SEQ ID NO.1, the light chain amino acid sequence of AS-MAB02 is shown in SEQ ID NO.2, the light chain amino acid sequence of AS-MAB03 is shown in SEQ ID NO.3, and the light chain amino acid sequence of AS-MAB04 is shown in SEQ ID NO.4; the heavy chain amino acid sequence of AS-MAB01 is shown in SEQ ID NO.5, the heavy chain amino acid sequence of AS-MAB02 is shown in SEQ ID NO.6, the heavy chain amino acid sequence of AS-MAB03 is shown in SEQ ID NO.7, and the heavy chain amino acid sequence of AS-MAB04 is shown in SEQ ID NO.8.

[0030] The optimal antibody pair was found to be: AS-MAB01 and AS-MAB02.

[0031] In the second step, the ruthenium-labeled monoclonal detection antibody AS-MAB02 reagent is prepared;

[0032] The third step involves using an electrochemiluminescence immunoassay analyzer for detection.

[0033] Step 1: Coat the monoclonal antibody reagent AS-MAB01 in a microplate at a concentration of 2.0–10.0 ug / mL and incubate at 4°C for 16–24 hours.

[0034] Step 2: Wash three times with Tween 20 phosphate buffer, drain the residual liquid, and then block with 1% Casein at room temperature for 1-2 hours.

[0035] Step 3: Prepare the samples, quality control samples, and test samples for fitting the standard curve;

[0036] Use serum to prepare standard curve samples and quality control samples. Dilute the test samples with sample diluent, with a minimum dilution factor of 1 to 100, to obtain standard curve samples, quality control samples and test samples.

[0037] Step 4: Add the sample used for fitting the standard curve, the quality control sample, and the test sample to the microplate, 100 μL / well, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST.

[0038] Step 5: Add 100 μL of ruthenium-labeled monoclonal detection antibody AS-MAB02 reagent to the microplate, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST.

[0039] Step 6: Add 100 μL of tri-n-propylamine, a co-reactant, to the microplate and read the results using an electrochemiluminescence immunoassay analyzer.

[0040] The beneficial effect of this disclosure is that, using the prepared semaglutide monoclonal antibody pair as the key reagent, a quantitative analytical method for semaglutide based on electrochemiluminescence immunoassay has been established. This method features high sensitivity, high specificity, and high throughput detection, and is applicable to the detection of semaglutide in various biological matrices such as human serum, monkey serum, or mouse serum, making its application very broad. The successful establishment of this method provides an innovative detection approach for the preclinical and clinical detection of semaglutide blood concentrations.

[0041] The kit has high sensitivity and can well meet the needs of blood drug concentration detection in clinical / preclinical biological samples.

[0042] The detection method can be used for the quantitative detection of semaglutide in serum samples from mice, monkeys, and humans. It can be used for the analysis of semaglutide blood concentrations in clinical / preclinical studies, providing reliable data support for semaglutide pharmacokinetic studies.

[0043] Further features and aspects of the present invention will be clearly described in the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0044] Figure 1 This is the result of antibody pairing S / N value;

[0045] Figure 2 This is a result of specificity verification;

[0046] Figure 3 This is a schematic diagram of the detection process;

[0047] Figure 4 It is a standard curve. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] 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.

[0050] The analytical method for detecting semaglutide content based on electrochemiluminescence immunoassay mainly includes the following steps:

[0051] The first step is the preparation of monoclonal antibodies and the screening of antibody pairs.

[0052] Step 1: Mice were immunized with the semaglutide standard conjugated to the carrier protein hemocyanin (KLH). Serum from immunized mice was collected for immunogenicity detection and titer analysis. Mice with the best immunogenicity were selected, and their spleen cells were fused with myeloma cells. Four monoclonal antibodies that specifically bind to semaglutide but do not cross-react with recombinant human GLP-1 were screened.

[0053] The light chain amino acid sequences of the four monoclonal antibodies are as follows: The light chain amino acid sequence of AS-MAB01 is SEQ ID NO.1:

[0054] DIQLTQTPLTLSTSVGERVSLTCKSSQSLIYDNYGVFQRPGQSPKRLIYLVSKLDSGVPKRFTGSGYGYDFSLKISNVQGEDFADYYCQQYCSFPYTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0055] The light chain amino acid sequence of AS-MAB02 is SEQ ID NO.2:

[0056] DIQLTQSPAPLSVTIGQPASLTCKASESLLYSNGKTYLNWLFQRPGQFPKRLIKAASNQCSGVPARFTGGYSGTDYTLTISNVETDDTAVYFCVQYNSTPRTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0057] The light chain amino acid sequence of AS-MAB03 is SEQ ID NO.3:

[0058] DIVLTQSPATLSVTPGDSVSLSCRASQSVYKYVAWHGHEIGQSPLYIKYISQSIDSGVPDRFTGSGAGTDYSLIIGSLESEDLGVYYCVQGYSFPLIFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0059] The light chain amino acid sequence of AS-MAB04 is SEQ ID NO.4:

[0060] DIVLTQSQKFLTATPGGDRSVTCRASQSVDNYNGKTYLNWIAQLFGQPPRLLFYLISKASSGVPDRFIGSGDGTDFTLKIGHVQSEDFGHYPCLQGTHFPLTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0061] The heavy chain amino acid sequences of the four monoclonal antibodies are as follows: The heavy chain amino acid sequence of AS-MAB01 is SEQ ID NO. 5:

[0062] EVTLQQSGAELAKPGASVKMTCTVSGFIQDTSGHNINWIVQRPEQGLEWIGNIDPANYTEYDQNFKDKATLTADKSSSTIFMQLSSLTTIDSAIYYCARAYIIFMRYSNIGVYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0063] The heavy chain amino acid sequence of AS-MAB02 is SEQ ID NO.6:

[0064] QVQLVISGKELAKPGTSVKMTCRAAGYTFTSDFYNWVKQRPAHGLEWIGDIYPGGGYPNYDPKFLMKGTISADTSSKTAFMQLSSLQSEDSAVYYCARGAYRAGIFYWGQGTLVTLSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK。

[0065] The heavy chain amino acid sequence of AS-MAB03 is SEQ ID NO.7:

[0066] DVQLKESGTELVKPGTSLRLSCKASGFTFSGSIMGVGWIRQSPAHGLEWIGHINSDGSTNYSNEKFQGATLTVDNSSKTVFLQVFSLTSEDSAVYYCARIAYSGVRIDGFTDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0067] The heavy chain amino acid sequence of AS-MAB04 is SEQ ID NO.8:

[0068] EVQLQESGAELIKPGASLRLSCKASGFNIQDSYVHWMKQRPEQGLEWIGWINPSNGSINYNEKFKSKATLTVDNDKSTHYLQLSSLTSEDTAVYYCARIIYYDGFDYWGQGTL VTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPC PPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIER TISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK.

[0069] Antibody titer is an important indicator of the binding ability of specific antibodies to antigens. This experiment used an indirect ELISA method to assess the titers of four monoclonal antibodies. The evaluation criteria were: wells with a signal value ≥2.0 (P / N ≥ 2.0) higher than the blank value for different dilutions of the test antibody were considered positive, and wells with a P / N < 2.0 were considered negative. The highest positive result was taken as the antibody titer. The specific procedure was as follows: Smegglutinin was diluted to 2.0 μg / mL and incubated overnight at 4°C in a 96-well ELISA plate. The next day, after washing and blocking, different dilutions of the test antibody and blank control samples were added and incubated with shaking. Then, HRP-labeled secondary antibody was added at a dilution of 1:8000 for co-reaction. Finally, TMB chromogenic solution was added for color development. After termination, the titers were measured at 450 nm and 630 nm using an ELISA reader, and the results were calculated. The experimental results are shown in Table 1 below.

[0070] Table 1: Results of Monoclonal Antibody Titer Evaluation

[0071]

[0072] According to the experimental results, the titer of AS-MAB01 can reach 1:50000, the titers of AS-MAB02 and AS-MAB04 can reach 1:150000, and the titer of AS-MAB03 is 1:10000.

[0073] Step 2: Then, using the ELISA method, each monoclonal antibody and its corresponding HRP-labeled antibody were paired. The S / N ratio was compared, and the pair with the highest S / N ratio was selected as the optimal antibody pair. The optimal antibody pair selection results are shown below. Figure 1 As shown, the optimal antibody pair is: AS-MAB01, AS-MAB02.

[0074] The selected specific monoclonal antibody pairs are both murine IgG kappa light chain antibodies. Both antibodies have high affinity, excellent sensitivity and strong specificity after pairing, and their tolerance to GLP-1 levels far exceed the normal physiological level of the human body, which can meet the needs of clinical testing.

[0075] Step 3: Perform specificity verification on the antibody pair.

[0076] Smegglutinin is a GLP-1 analogue with 94% homology to the amino acid sequence of natural GLP-1. In healthy individuals, the GLP-1 concentration ranges from approximately 12.0 to 42.0 ng / mL. This study assessed the specificity of this method by adding GLP-1 levels far exceeding normal physiological levels (200.0 ng / mL, 100.0 ng / mL, 50.0 ng / mL, and 25.0 ng / mL) to a biological matrix to simulate endogenous substances that may be present during sample detection. The experimental results are as follows: Figure 2 As shown, the ECL value of samples containing GLP-1 was close to the blank value, and there was no dose-dependent effect, indicating that the antibody is highly specific for semaglutide, even though it does not recognize GLP-1 in the sample.

[0077] The second step is to prepare the ruthenium-labeled monoclonal detection antibody AS-MAB02 reagent.

[0078] Ruthenium labeling for antibody detection The SULFO-Ru Labeling kit was used, and the experimental procedure was strictly followed according to the instructions. The specific labeling procedure is as follows: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide sodium salt (Sulfo-NHS) were diluted to 50.0 mg / mL working solutions using 2-morpholine ethanesulfonate buffer (MES), and Ru lyophilized powder was dissolved to a working solution of 10.0 mg / mL. The Ru activation working solution was prepared by mixing Ru:Sulfo-NHS:EDC at a ratio of 100:15:15 (V / V / V) and incubating at 25°C with shaking in the dark for 25 min to activate Ru. The antibody to be labeled, AS-MAB02, and the Ru activation working solution were mixed at a 1:1 (mol / mol) and incubated at 25°C with shaking in the dark for 2 h. After labeling, the mixture was purified by ultrafiltration.

[0079] The third step involves using an electrochemiluminescence immunoassay analyzer for detection.

[0080] Step 1: Coat the monoclonal antibody reagent AS-MAB01 in a microplate at a concentration of 2.0–10.0 ug / mL and incubate at 4°C for 16–24 hours.

[0081] The preferred concentration is 5.0 μg / mL. The preferred incubation time is 18-20 hours.

[0082] Step 2: Wash three times with Tween 20 phosphate buffer (PBST, Tween 20 content is 0.05%), drain the residual liquid, and block with 1% Casein at room temperature for 1-2 hours, preferably 1 hour.

[0083] Step 3: Prepare the samples, quality control samples, and test samples for fitting the standard curve.

[0084] Standard curve samples and quality control samples are prepared using a biological matrix (human, monkey, or mouse serum). The test samples are diluted with sample diluent, with a minimum dilution factor (MRD) of 1 to 100, to obtain standard curve samples, quality control samples, and test samples.

[0085] Step 4: Add the sample used for fitting the standard curve, the quality control sample, and the test sample to the microplate at 100 μL / well, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST.

[0086] Step 5: Add 100 μL of ruthenium-labeled monoclonal detection antibody AS-MAB02 reagent to the microplate, incubate at 500 rpm for 1 hour at room temperature, and wash three times with PBST.

[0087] Step 6: Add 100 μL of tri-n-propylamine (1×Read Buffer) as a co-reactant to the microplate, and read the results using an electrochemiluminescence immunoassay analyzer.

[0088] To reflect the relationship between the analyte concentration and the analytical platform response, a stable and reliable standard curve was established for this analytical method. A series of concentration gradients were prepared using semaglutide standards, with standard curve points including 200.0, 100.0, 25.0, 5.0, 1.0, 0.25, and 0.1 ng / mL, a blank point, and anchor points of 1, 200.0, and 0.05 ng / mL. The sensitivity of the detection method was 0.1 ng / mL. A representative standard curve is shown below. Figure 4 As shown in Table 2, the percentage of the coefficient of variation (%CV) and the percentage of the relative error (%RE) of the back-calculated concentrations at each concentration point are both within 10%.

[0089] Table 2: Standard Curve Test Results

[0090]

[0091] The reagent sensitivity was determined by the limit of detection (LOB) of the detection method, which was performed according to the following experimental method. Twenty tests were conducted on the zero-concentration calibrator. The concentration ECL values ​​of the 20 measured results were compared with the ECL values ​​of adjacent concentration calibrators (0.02 ng / mL). A two-point regression was performed to derive a linear equation. The ECL value corresponding to M+2SD was substituted into the equation to calculate the corresponding concentration, which is the LOB. The results are shown in Table 3. The LOB of this detection method is 0.0016 ng / mL.

[0092] Table 3: Sensitivity Test Results of Detection Method

[0093]

[0094] This invention involves conducting multiple independent analytical batches across different days, on the same electrochemical microplate, and on different microplates. Each batch includes one standard curve and three sets of 5-level QCs, with a ULOQ of 200.0 ng / mL, a High Quality Control (HQC) of 150.0 ng / mL, a Moderate Quality Control (MQC) of 5.0 ng / mL, a Low Quality Control (LQC) of 0.3 ng / mL, and an LLOQ of 0.1 ng / mL. Each sample is replicated in two wells. The validation results are shown in Table 4. The inter-batch %CV for each QC is ≤15.0%, and |%RE| is ≤10.0%, indicating that the detection method has good precision and accuracy.

[0095] Table 4: Precision and Accuracy Verification Results

[0096]

[0097] Species inspection and examination:

[0098] According to the experimental procedure, a series of concentration gradients (200.0, 100.0, 25.0, 5.0, 1.0, 0.25, 0.1 ng / mL) of semaglutide standard were prepared using different biological matrices (human serum, monkey serum, or rat serum) to fit a standard curve, and the %CV and %RE of the back-calculated concentration were calculated. The experimental results are shown in Table 5. In various biological matrices, the %CV and %RE of the back-calculated concentration of semaglutide were ≤20.0% and ≤20.0%, respectively. These results indicate that this method can be used for the detection of semaglutide in human serum, monkey serum, and rat serum, and has a wide range of applications.

[0099] Table 5: Cross-species investigation

[0100]

[0101] As can be seen, a quantitative analytical method for semagravirtuous liposome based on electrochemiluminescence immunoassay was established using the prepared semagravirtuous liposome monoclonal antibody pair as the key reagent. This method features high sensitivity, high specificity, and high throughput detection, and is applicable to the detection of semagravirtuous liposome in various biological matrices, including human serum, monkey serum, and mouse serum, making its application very broad. The successful establishment of this method provides an innovative detection approach for the preclinical and clinical blood concentration determination of semagravirtuous liposome.

Claims

1. A monoclonal antibody pair for detecting semaglutide, characterized in that, The invention comprises monoclonal antibodies AS-MAB01 and AS-MAB02. The light chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.

5. The light chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.2, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.

6.

2. A kit for detecting semaglutide, characterized in that, This includes monoclonal antibody pairs for detecting semaglutide, washing buffer, labeling reagents, electrochemiluminescence microplates, and electrochemiluminescence substrate solutions; The monoclonal antibody pair for detecting semaglutide comprises monoclonal antibody AS-MAB01 and monoclonal antibody AS-MAB02. The light chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB01 is shown in SEQ ID NO.

5. The light chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.2, and the heavy chain amino acid sequence of monoclonal antibody AS-MAB02 is shown in SEQ ID NO.

6.

3. The kit for detecting semaglutide according to claim 2, characterized in that, The labeling reagent is a ruthenium labeling reagent.

4. The kit for detecting semaglutide according to claim 2, characterized in that, The washing buffer is Tween 20 phosphate buffer.

5. The kit for detecting semaglutide according to claim 2, characterized in that, The kit for detecting smegglutinin also includes a sample diluent.

6. The kit for detecting semaglutide according to claim 5, characterized in that, The sample diluent was Casein buffer.

7. The kit for detecting semaglutide according to claim 6, characterized in that, The Casein buffer solution contains 0.01% to 0.5% Tween 20.

8. The kit for detecting semaglutide according to claim 2, characterized in that, The electrochemiluminescent substrate solution is an electrochemiluminescent substrate solution containing tri-n-propylamine.

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