Kit for detecting simeglutide neutralizing antibody and application thereof

Abstract

The invention provides a kit for detecting a stemeglutide neutralizing antibody and application of the kit. The kit comprises recombinant human GLP-1R receptor protein, a standard rabbit anti-stemeglutide polyclonal antibody, biotin labeled stemeglutide and HRP-streptavidin. If the neutralizing antibody is combined with biotin-labeled simeglutide, the simeglutide drug cannot be combined with GLP-1R, the added HRP-streptavidin cannot be connected later, and finally the added HRP substrate TMB cannot be catalyzed to realize color development, and the method can be used for qualitatively and quantitatively judging whether a patient needs to change a medication strategy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a kit for detecting smaglutide neutralizing antibodies and applications thereof. Background Art

[0002] Semaglutide is a novel, long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA). Its structure is similar to native human glucagon-like peptide-1 (GLP-1), sharing up to 94% amino acid sequence identity. It is used to control blood sugar in adults with type 2 diabetes. Semaglutide acts through the GLP-1 receptor, selectively binding to and activating it, stimulating insulin secretion and reducing glucagon secretion to lower blood sugar. Compared to traditional GLP-1RAs, its half-life is extended to 7 days.

[0003] The structural formula of smaglutide is shown in Figure 1 After long-term semaglutide infusion, some users experience diminished or even absent efficacy. This is because the body produces autoimmune antibodies against semaglutide, preventing it from binding to the GLP-1R. In particular, neutralizing antibodies generated at the N-terminus of the peptide can largely inhibit binding between the peptide and the receptor. Therefore, to determine if this neutralizing antibody production is responsible for the loss of efficacy, a method to detect these antibodies is needed. Summary of the Invention

[0004] In view of this, the present invention provides a kit for detecting neutralizing antibodies against smaglutide and its application, which are used to identify the reasons why the efficacy of smaglutide is lost.

[0005] The technical solution of the present invention is achieved as follows: On the one hand, the present invention provides a kit for detecting neutralizing antibodies against smaglutide, comprising a recombinant human GLP-1R receptor protein, a standard rabbit anti-smaglutide polyclonal antibody, biotin-labeled smaglutide, and HRP-streptavidin.

[0006] Based on the above technical solution, preferably, the recombinant human GLP-1R receptor protein includes amino acids 24-145 of the human GLP-1R receptor protein, and a Flag tag is added to its N-terminus.

[0007] The amino acid sequence of human GLP-1R receptor protein (glucagon-like peptide-1 receptor, UniProt number: P43220) at positions 24-145 is: RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLF CNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLY.

[0008] Based on the above technical solution, preferably, the preparation method of the rabbit anti-smaglutide polyclonal antibody is: artificially synthesizing the N-terminal 20 amino acid fragment of smaglutide (HAEGTFTSDVSSYLEGQAAK), coupling it with KLH protein to prepare an immunogen, and then immunizing rabbits to obtain the polyclonal antibody.

[0009] Smaglutide is a GLP-1 analog, and the N-terminal region of GLP-1 (glucagon-like peptide-1) is the key region for its binding to the GLP-1 receptor, so this part of the sequence is usually highly antigenic.

[0010] Furthermore, a 20-amino acid length is within the conventional range for peptide antigen design. It is short enough for easy chemical synthesis and coupling to carrier proteins (such as KLH), yet long enough to cover multiple immune epitopes, thereby enhancing immunogenicity and antibody recognition. Therefore, the present invention selected a 20-amino acid fragment at the N-terminus, which exhibits both high antigenicity and enhanced antibody recognition.

[0011] On the basis of the above technical solution, preferably, the kit further comprises an ELISA plate, a washing solution, a color developer and a stop solution.

[0012] Based on the above technical solution, preferably, the ELISA plate is a 48-well or 96-well ELISA plate, the washing solution is a PBS buffer containing 0.01%-0.05% v / v Tween-20, the color developer is TMB, and the stop solution is 1-2 mol / L sulfuric acid.

[0013] On the other hand, the present invention also provides use of the kit in detecting smaglutide neutralizing antibodies.

[0014] On the basis of the above technical solution, preferably, the detection method includes the following steps: S1, immobilize the recombinant human GLP-1R receptor protein on the wells of the ELISA plate; S2, adding biotin-labeled smaglutide and rabbit anti-smaglutide polyclonal antibody or the test sample to the wells of the ELISA plate in step S1, incubating to allow the biotin-labeled smaglutide to compete with the recombinant human GLP-1R receptor protein in the wells, and then washing the wells; S3, adding HRP-streptavidin to the wells of the ELISA plate in step S2, incubating, and then washing the wells with a washing solution; S4, adding a color developing agent to the wells of the ELISA plate in step S3, incubating, and then stopping the reaction with a stop solution and detecting the color intensity.

[0015] Based on the above technical solution, preferably, the method for fixing the recombinant human GLP-1R receptor protein to the wells of the ELISA plate is: S11, coating protein: dilute the recombinant human GLP-1R receptor protein with diluent and add it to the wells of the ELISA plate and incubate; S12, plate washing: After the incubation is completed, the coating solution is discarded and the plate is washed with washing solution; S13, blocking: add blocking solution to each well and incubate. After incubation, pour out the blocking solution and wash the plate.

[0016] Based on the above technical solution, preferably, the diluent is a PBS buffer containing 0.1%-1% w / v BSA and 0.02%-0.05% v / v pc300 preservative, and the blocking solution is a PBS buffer containing 0.5%-1% wt / v BSA.

[0017] Based on the above technical solutions, preferably, the sample to be tested is whole blood, serum or plasma.

[0018] The kit for detecting neutralizing antibodies against smaglutide and its application of the present invention have the following beneficial effects compared with the prior art: If the neutralizing antibody of smaglutide binds to biotin-labeled smaglutide, the smaglutide drug will be unable to bind to GLP-1R, and will not be able to connect to the added HRP-streptavidin, and ultimately will not be able to catalyze the added HRP substrate TMB to achieve color development. Therefore, this method is a competitive method.

[0019] If neutralizing antibodies to smaglutide are present in human blood, particularly those that bind within the N-terminal 20 amino acids, competition will occur, resulting in a lighter color. Finally, the approximate antibody concentration can be calculated by fitting the sample OD value with the OD of the calibration curve. This method can be used for both qualitative and quantitative analysis to determine whether a patient's medication strategy needs to be changed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a standard curve diagram of an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] The HRP-streptavidin (SABC) used in the present invention was obtained from Wuhan Fine Biotechnology Co., Ltd.

[0024] The recombinant human GLP-1R receptor protein used in the present invention includes amino acids 24-145 of the human GLP-1R receptor protein, and a Flag tag is added to the N-terminus.

[0025] The amino acid sequence of positions 24-145 of the human GLP-1R receptor protein (UniProt number: P43220) is: RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDG EPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLY.

[0026] The preparation method of the rabbit anti-smaglutide polyclonal antibody used in the present invention is as follows: artificially synthesizing the N-terminal 20 amino acid fragment of smaglutide (HAEGTFTSDVSSYLEGQAAK), coupling it to KLH protein to prepare an immunogen, and then immunizing rabbits (primary immunization: immunogen + Freund's complete adjuvant; booster immunization: immunogen + Freund's incomplete adjuvant (14th, 28th, 42nd day), collecting serum after immunization, and purifying antibodies) to obtain the polyclonal antibody.

[0027] The biotin-labeled smaglutide labeling method used in the present invention is as follows: smaglutide is dissolved in 0.1 M NaHCO3 buffer (pH 8.0) to 5 mg / mL; NHS-activated biotin is dissolved in DMF to prepare a 10 mM stock solution; 10 μL of the stock solution is slowly added to the smaglutide solution and mixed in the dark; the mixture is reacted at 4°C for 2 hours, and unbound biotin is removed by ultrafiltration.

[0028] Example 1 This embodiment provides a method for detecting smaglutide neutralizing antibodies, comprising the following steps: S1, fix the recombinant human GLP-1R receptor protein to the wells of a 48T (8 wells × 6 strips) ELISA plate Coating protein: Dilute recombinant human GLP-1R receptor protein to 4 µg / mL in diluent (PBS buffer, pH 7.4, containing 0.1% w / v BSA and 0.05% v / v PC300 preservative). Add 100 µL of protein coating solution to each well and gently tap the plate to avoid air bubbles. Incubate at 37°C for 2 h.

[0029] Wash the plate: Pour off the coating solution and fill each well with PBST detergent (0.01% v / v Tween-20 in PBS buffer, pH 7.4). Let it stand for 1 minute, then invert and pat dry. Repeat the wash three times.

[0030] Blocking: Add 200 µL of blocking solution (PBS buffer containing 1% wt / v BSA) to each well. Gently tap to mix and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate three times with PBST, soaking for 1 minute each time.

[0031] S2. Add 50 µL / well of biotin-labeled smaglutide (1 µg / ml) and 50 µL / well of rabbit anti-smaglutide polyclonal antibody (10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0 ng / ml) at different concentrations to the ELISA plate wells of step S1. Gently tap the plate to mix for 1 min to ensure sufficient mixing. Then incubate at 37°C for 45 min to allow it to compete with the recombinant human GLP-1R receptor protein in the wells. Then wash the wells three times with PBST, soaking for 1 min each time.

[0032] S3, add 100 μL / well of HRP-streptavidin to the wells of the ELISA plate in step S2, seal the plate and incubate at 37°C for 30 minutes, then wash the plate 5 times with PBST, soaking for 1 minute each time.

[0033] In step S4, add 90 μL / well of TMB colorimetric reagent to the wells of the ELISA plate prepared in step S3, seal the plate, and incubate at 37°C for 10 minutes. After incubation, add 50 μL of stop solution (1 mol / L sulfuric acid) to each well to terminate the reaction. Immediately read the absorbance at 450 nm to measure the color intensity and calculate the color intensity.

[0034] Example 2 S1, fix the recombinant human GLP-1R receptor protein to the wells of 96T (8 wells × 12 strips) ELISA plate Coating protein: Dilute recombinant human GLP-1R receptor protein to 4 µg / mL in diluent (PBS buffer, pH 7.4, containing 1% w / v BSA and 0.02% v / v PC300 preservative). Add 100 µL of protein coating solution to each well and gently tap the plate to avoid air bubbles. Incubate at 37°C for 2 h.

[0035] Wash the plate: Pour off the coating solution and fill each well with PBST detergent (0.05% v / v Tween-20 in PBS buffer, pH 7.4). Let it stand for 1 minute, then invert and pat dry. Repeat the wash three times.

[0036] Blocking: Add 200 µL of blocking solution (PBS buffer containing 0.5% wt / v BSA) to each well. Gently tap to mix and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate three times with PBST, soaking for 1 minute each time.

[0037] S2. Add 50 µL / well of biotin-labeled smaglutide (1 µg / ml) and 50 µL / well of rabbit anti-smaglutide polyclonal antibody (10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0 ng / ml) at different concentrations to the ELISA plate wells of step S1. Gently tap the plate to mix for 1 min to ensure sufficient mixing. Then incubate at 37°C for 45 min to allow it to compete with the recombinant human GLP-1R receptor protein in the wells. Then wash the wells three times with PBST, soaking for 1 min each time.

[0038] S3, add 100 μL / well of HRP-streptavidin to the wells of the ELISA plate in step S2, seal the plate and incubate at 37°C for 30 minutes, then wash the plate 5 times with PBST, soaking for 1 minute each time.

[0039] In step S4, add 90 μL / well of TMB colorimetric reagent to the wells of the ELISA plate prepared in step S3, seal the plate, and incubate at 37°C for 10 minutes. After incubation, add 50 μL of stop solution (2 mol / L sulfuric acid) to each well to terminate the reaction. Immediately read the absorbance at 450 nm to measure the color intensity and calculate the color intensity.

[0040] Example 3 S1, fix the recombinant human GLP-1R receptor protein to the wells of a 48T (8 wells × 6 strips) ELISA plate Coating protein: Dilute recombinant human GLP-1R receptor protein to 4 µg / mL in diluent (PBS buffer, pH 7.4, containing 0.5% w / v BSA and 0.04% v / v PC300 preservative). Add 100 µL of protein coating solution to each well and gently tap the plate to avoid air bubbles. Incubate at 37°C for 2 h.

[0041] Wash the plate: Pour off the coating solution and fill each well with PBST detergent (0.04% v / v Tween-20 in PBS buffer, pH 7.4). Let it stand for 1 minute, then invert and pat dry. Repeat the wash three times.

[0042] Blocking: Add 200 µL of blocking solution (PBS buffer containing 0.8% wt / v BSA) to each well. Gently tap to mix and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate three times with PBST, soaking for 1 minute each time.

[0043] S2. Add 50 µL / well of biotin-labeled smaglutide (1 µg / ml) and 50 µL / well of rabbit anti-smaglutide polyclonal antibody (10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0 ng / ml) at different concentrations to the ELISA plate wells of step S1. Gently tap the plate to mix for 1 min to ensure sufficient mixing. Then incubate at 37°C for 45 min to allow it to compete with the recombinant human GLP-1R receptor protein in the wells. Then wash the wells three times with PBST, soaking for 1 min each time.

[0044] S3, add 100 μL / well of HRP-streptavidin to the wells of the ELISA plate in step S2, seal the plate and incubate at 37°C for 30 minutes, then wash the plate 5 times with PBST, soaking for 1 minute each time.

[0045] In step S4, add 90 μL / well of TMB colorimetric reagent to the wells of the ELISA plate prepared in step S3, seal the plate, and incubate at 37°C for 10 min. After incubation, add 50 μL of stop solution (1.5 mol / L sulfuric acid) to each well to terminate the reaction. Immediately read the absorbance at 450 nm to determine the color intensity and calculate the results. See Table 1.

[0046] Table 1 Absorbance of Examples Figure 1 is the curve of smaglutide neutralizing antibody concentration and absorbance, and the linear correlation coefficient R 2 =0.99604, which is greater than 0.990, indicating that the kit and the detection method of the present invention have high accuracy and provide an effective tool for the quantitative detection of smaglutide neutralizing antibodies.

[0047] Example 4 Detection of patient samples and ordinary people's samples In this example, the detection method of Example 1 was used to detect patient samples and samples from ordinary people. The samples from different patients were diluted 1000 times, and the samples from ordinary people were diluted twice for testing. The results are shown in Table 2. The patient samples and the samples from ordinary people were obtained from the cooperative unit, Tongji Hospital of Huazhong University of Science and Technology.

[0048] Table 2 Test results As shown in Table 2, the kit and detection method of the present invention can detect smaglutide neutralizing antibodies in patients, and the concentration can be used to determine whether the patient needs to change the medication strategy.

[0049] Example 5 Detection recovery rate 5 μg / mL of semaglutide polyclonal antibody was added to the serum sample and diluted 1000 times, and the test was performed using the method of Example 1 (partial data). The results are shown in Table 3.

[0050] Table 3 Test results The following data are calculated in summary: Table 4 Recovery rate As shown in Table 3, the kit and detection method of the present invention can be used for serum, EDTA plasma and heparin plasma, and the recovery rate reaches above 97%, which is within the qualified recovery range of 80%-120% (universal standard).

[0051] Linearity: 10 μg / mL semaglutide antibody was diluted 1000 times to a concentration of 10 mg / mL, and then diluted 2-fold, 4-fold, and 8-fold, respectively, and tested using the method of Example 1 (partial data). The results are shown in Table 5 Table 5 Test results The summary calculations yield the following results: Table 6 Recovery rates of different test samples As shown in Table 3, the kit and detection method of the present invention have good linear effects. The recovery rates of samples at different dilution ratios are all high and are within the qualified recovery range of 80%-120% (universal standard).

[0052] Stability: The unopened kit (using Example 1 as an example) was subjected to stability tests at 37°C and 2-8°C.

[0053] Table 7 Kit stability As shown in Table 7, the recovery rate of the kit of the present invention can still reach 95%-100% when stored at 2-8°C for 6 months, and the recovery rate can reach 80% when stored at 37°C for 1 month, indicating that the stability of the kit is good.

[0054] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for detecting neutralizing antibodies to smaglutide, characterized in that: Includes recombinant human GLP-1R receptor protein, standard rabbit anti-smaglutide polyclonal antibody, biotin-labeled smaglutide and HRP-streptavidin.

2. A kit for detecting neutralizing antibodies to smaglutide according to claim 1, characterized in that: The recombinant human GLP-1R receptor protein includes amino acids 24-145 of the human GLP-1R receptor protein, and a Flag tag is added to the N-terminus.

3. A kit for detecting neutralizing antibodies to smaglutide according to claim 1, characterized in that: The preparation method of the rabbit anti-smaglutide polyclonal antibody comprises the following steps: artificially synthesizing the 20 amino acid fragments at the N-terminus of smaglutide, coupling it with KLH protein to prepare an immunogen, and then immunizing rabbits to obtain the polyclonal antibody.

4. A kit for detecting neutralizing antibodies against smaglutide according to claim 1, characterized in that: The kit also includes an enzyme labeling plate, a washing solution, a color developing agent and a stop solution.

5. A kit for detecting neutralizing antibodies against smaglutide according to claim 4, characterized in that: The ELISA plate is a 48-well or 96-well ELISA plate, the washing solution is a PBS buffer solution containing 0.01%-0.05% v / v Tween-20, the color developer is TMB, and the stop solution is 1-2 mol / L sulfuric acid.

6. Use of the kit according to any one of claims 1 to 5 in detecting neutralizing antibodies against smaglutide.

7. The use according to claim 6, characterized in that: The detection method includes the following steps: S1, immobilize the recombinant human GLP-1R receptor protein on the wells of the ELISA plate; S2, adding biotin-labeled smaglutide and rabbit anti-smaglutide polyclonal antibody or the test sample to the wells of the ELISA plate in step S1, incubating to allow the biotin-labeled smaglutide to compete with the recombinant human GLP-1R receptor protein in the wells, and then washing the wells; S3, adding HRP-streptavidin to the wells of the ELISA plate in step S2, incubating, and then washing the wells with a washing solution; S4, adding a color developing agent to the wells of the ELISA plate in step S3, incubating, and then stopping the reaction with a stop solution and detecting the color intensity.

8. The use according to claim 7, characterized in that: The method for fixing the recombinant human GLP-1R receptor protein to the wells of the ELISA plate is as follows: S11, coating protein: dilute the recombinant human GLP-1R receptor protein with diluent and add it to the wells of the ELISA plate and incubate; S12, plate washing: After the incubation is completed, the coating solution is discarded and the plate is washed with detergent; S13, blocking: add blocking solution to each well and incubate. After incubation, pour out the blocking solution and wash the plate.

9. The use according to claim 8, characterized in that: The diluent is a PBS buffer solution containing 0.1%-1% w / v BSA and 0.02%-0.05% v / v pc300 preservative, and the blocking solution is a PBS buffer solution containing 0.5%-1% wt / v BSA.

10. The use according to any one of claims 6 to 9, characterized in that: The sample to be tested is whole blood, serum or plasma.

Images