Monoclonal antibody of humanized anti-ATR001 oligopeptide antigen, antibody quantitative detection kit and application

Abstract

The invention provides a monoclonal antibody of a humanized anti-ATR001 oligopeptide antigen, an antibody quantitative detection kit and application. The monoclonal antibody comprises a heavy chain and a light chain of a specific amino acid sequence, and the kit comprises a coated plate coated with an ATR001 oligopeptide antigen, a standard substance, a matched diluent, a horse radish peroxidase labeled goat anti-human IgG antibody, a developing solution, a stop solution and a concentrated washing solution. The ATR001 oligopeptide antigen is moderately modified, so that the ATR001 oligopeptide antigen has higher coating stability and easiness in coupling, and the sensitivity and the specificity of a detection method can be remarkably improved. The kit disclosed by the invention can be used for detecting the level of the anti-ATR001 oligopeptide antibody generated in human serum after subcutaneous injection of the ATRQbeta-001 therapeutic antihypertensive vaccine, so that an immune response and immunogenicity evaluation method is provided for clinical application of the vaccine.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic reagents, specifically to a humanized monoclonal antibody against the ATR001 short peptide antigen, an antibody quantitative detection kit, and its application. Background Technology

[0002] Hypertension is a major public health problem worldwide. The pathogenesis of hypertension is complex, involving the sympathetic nervous system, the renin-angiotensin system (RAS), and the immune system. In 1999, Wallukat et al. discovered an anti-angiotensin II receptor type 1 extracellular second-cyclic peptide autoantibody (AT1R-ECL2 165–191: IHRNVF-FI-INTNITVCAFHYESQNSTL) in the serum of patients with preeclampsia, and experimental studies confirmed its agonist-like effect. Liao et al. detected anti-AT1R-ECL2 antibodies in the serum of patients with refractory hypertension. In a 3.3-year follow-up of hypertensive stroke patients, they found that patients with positive anti-AT1R-ECL2 antibodies had an increased stroke recurrence rate and mortality. Clinical trial results of SOT-AT1 demonstrated that for hypertensive patients with positive anti-AT1R-ECL2 antibodies, angiotensin receptor blockers (ARBs) were more effective than angiotensin-converting enzyme inhibitors (ACEIs) in lowering blood pressure. These clinical studies demonstrate that anti-AT1R-ECL2 antibodies have an agonist-like effect.

[0003] Thinking in reverse, are antibodies targeting short peptides of the AT1R receptor agonists or blockers? Therefore, a research hypothesis for an AT1R therapeutic antihypertensive vaccine is proposed. Chinese invention patent application number 200610019290.X discloses the application of a human immunogenic peptide fragment of angiotensin II receptor type 1, whose short peptide vaccine not only effectively lowers SHR blood pressure but also significantly inhibits its cardiac and vascular remodeling. Chinese invention patent application number 201010028904.7 discloses a method for preparing Qβ-2aa phage virus-like particle protein and its uses, which constructs the phage virus-like particle capsid protein Qβ-2aa through improvements to the vaccine vector. Chinese invention patent application number 201110171715.X discloses an angiotensin II receptor type 1 polypeptide-carrier vaccine and its uses, which invented the ATRQβ-001 therapeutic antihypertensive vaccine. Further research revealed that monoclonal antibodies targeting the ATR001 short peptide have the function of biased regulation of the AT1R / β-arrestin2 signaling pathway, activating the RAS system without feedback (Anti-ATR001 monoclonal antibody ameliorates atherosclerosis through beta-arrestin2 pathway. Biochem Biophys Res Commun. 2021 Mar 12:544:1-7). Anti-ATR001 short peptide antibodies specifically target the 181-187 peptide antigenic epitopes of AT1R-ECL2, among which Phe182-His183-Tyr184 are key binding sites for ATR001 short peptide antibodies. After the anti-ATR001 short peptide monoclonal antibody specifically binds to AT1R, it does not compete with angiotensin II for receptors, does not affect the Gq and Gi signaling pathways, and does not affect renin release mediated by the AC5 / 6-cAMP pathway (Anti-ATR001 monoclonal antibody ameliorates atherosclerosis through beta-arrestin2 pathway. Biochem Biophys Res Commun. 2021 Mar 12:544:1-7).

[0004] The aforementioned monoclonal antibodies are designed for animals. During the clinical trials of this vaccine, there is an urgent need to develop a quantitative detection kit for human anti-ATR001 short peptide antibodies. It is necessary to develop a humanized monoclonal antibody against the anti-ATR001 short peptide antigen as a standard to establish a standard curve and positive control for the quantitative detection of anti-ATR001 short peptide antibodies. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a humanized monoclonal antibody against the ATR001 short peptide antigen, along with an antibody quantitative detection kit and its applications. This invention prepares a monoclonal antibody, which is then used to prepare an antibody quantitative detection kit and its applications. The peptide coating stability is further enhanced, and it is easy to conjugate. The prepared antibody detection kit exhibits higher sensitivity and stronger specificity. This kit can be used to detect anti-ATR001 short peptide antibodies generated by the immune response after subcutaneous injection of the HJY-ATRQβ-001 therapeutic antihypertensive vaccine in humans.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a humanized monoclonal antibody against the ATR001 short peptide antigen. The monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain includes three variable regions, namely heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and its amino acid sequence is as follows: The heavy chain CDR1 sequence is GYTFSEYTMH. The heavy chain CDR2 sequence is GIDPRNGGTTYTPKFKG. The heavy chain CDR3 sequence is YDYRIYYGMDY; The light chain includes three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3, with the following amino acid sequences: The light chain CDR1 sequence is RSSTGPVTTSNYAN. The light chain CDR2 sequence is GANIRAP. The light chain CDR3 sequence is ALWYSNHFI.

[0007] Furthermore, the amino acid sequence of the heavy chain is as follows: EVQLQQSGPELVKPGTSVKISCKTS GYTFSEYTMH WVKQSHGKSLEWIG GIDPRNGGTTYTPKFKG KATLTVDRSSSTAYMELRRSLTSEDSAVYYCVR YDYRIYYGMDY WGQGTSVTVSS; The amino acid sequence of the light chain is as follows: QAVVTQESALTTSPGETVTLTC RSSTGPVTTSNAYAN WVQEKPDHLFTGLIG GANIRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHFI FGSGTKVTVL.

[0008] This invention also provides the application of a humanized monoclonal antibody against the ATR001 short peptide antigen in the preparation of a detection kit for detecting the content of anti-ATR001 short peptide antibodies produced after subcutaneous injection of the ATRQβ-001 therapeutic antihypertensive vaccine into the human body.

[0009] The ATRQβ-001 therapeutic antihypertensive vaccine is disclosed in Chinese invention patent application number 201110171715.X, which discloses an angiotensin II receptor type 1 polypeptide-carrier vaccine and its use. After subcutaneous injection of the ATRQβ-001 therapeutic antihypertensive vaccine into the human body, anti-ATR001 short peptide antibodies will be generated in the serum.

[0010] The present invention also provides a quantitative detection kit for serum anti-ATR001 short peptide antibody, the detection kit comprising a coated plate coated with ATR001 short peptide antigen and a standard.

[0011] Furthermore, the amino acid sequence of the ATR001 short peptide antigen is shown in SEQ ID No. 1: CGGAFHYESQ.

[0012] Furthermore, the coated plate is formed by coupling the ATR001 short peptide antigen with the carrier protein bovine serum albumin and then coating it.

[0013] Furthermore, the standard contains the monoclonal antibody as described in claim 1 or 2, wherein the monoclonal antibody content in the standard is 640 ng / mL.

[0014] Furthermore, the test kit includes sample diluent, horseradish peroxidase-labeled goat anti-human IgG antibody, TMB chromogenic solution, stop solution, and 20× concentrated washing solution.

[0015] 20× concentrated washing buffer is PBST (phosphate buffer): 2 M PBS (prepared with NaCl, KCl, Na2HPO4, KH2PO4, pH 7.4), containing 10% TWEEN-20.

[0016] Furthermore, the diluent for the sample is a phosphate buffer containing bovine serum albumin; and the mass fraction of bovine serum albumin in the phosphate buffer is 1%.

[0017] The above-mentioned test kit is used for detecting anti-ATR001 short peptide antibodies. This test kit is used to detect anti-ATR001 short peptide antibodies, providing a detection method for the clinical application of the ATRQβ-001 therapeutic antihypertensive vaccine, and is also used to evaluate the immunogenicity and immune response effect of the ATRQβ-001 vaccine.

[0018] The method for detecting the concentration of serum anti-ATR001 short peptide antibody using the above kit includes the following steps: 1) Solution preparation: Dilute 50mL of 20× concentrated washing solution to 1× washing solution with distilled water or deionized water; 2) Prepare a gradient concentration of standards: Dilute the standards with sample diluent to concentrations of 200, 150, 100, 50, 25, 12.5, 6.25, and 0 ng / mL, respectively; 3) Sample addition: Dilute the sample to be tested with sample diluent, with a dilution ratio ranging from 1:10 to 1:10000; add the diluted standards of different concentrations and the sample to be tested to the corresponding wells (100 μL per well). Gently shake to mix. 4) Incubation: After sealing the plate with sealing film, incubate it in a 37℃ incubator for 60 minutes; 5) Washing: Carefully peel off the sealing film and wash the plate 5 times with a plate washer (or wash it by hand). Soak for 30 seconds each time. Fill each well with washing solution during washing. After the last wash, pat the plate dry on absorbent paper as much as possible. 6) Add enzyme: After shaking well, add 100 μL of horseradish peroxidase-labeled goat anti-human IgG antibody (which is an enzyme conjugate) to each well and gently shake to mix. 7) Incubation: After sealing the plate with another sheet of sealing film, place it in a 37°C incubator for 45 minutes; 8) Washing: Same as step 5.

[0019] 9) Color development: Add 100 μL TMB color developer to each well and develop at 37±1℃ in the dark for 8±1 minutes; 10) Measurement: Add 100 μL of stop solution to each well to stop the reaction, gently shake to mix, and measure the absorbance A value of each well at a wavelength of 450 nm-630 nm within 5 minutes. 11) Result Interpretation: Taking the concentration of the standard as x, the OD corresponding to the standard... 450 nm-630nm Given the value of y, perform a fourth-order polynomial fitting: Y = a + bX + cX^2 + dX^3 + eX^4 In the formula, Y is the absorbance value A corresponding to the standard, X is the concentration of the standard (ng / mL), and a, b, c, d, and e are all constants. Substitute the absorbance value of the sample well into the fitted equation to calculate the concentration x of the anti-ATR001 short peptide antibody in the sample well; calculate the concentration of the anti-ATR001 short peptide antibody in the sample to be tested based on the following formula; y = x * N; In the formula, y is the concentration of anti-ATR001 short peptide antibody in the sample to be tested, in ng / mL; x represents the concentration of anti-ATR001 short peptide antibody in the well of the coated plate, in ng / mL; N is the dilution factor.

[0020] This invention modifies the original ATR001 antigen polypeptide sequence to increase the stability, sensitivity, and specificity of the detection method. Compared with the original sequence, the modified polypeptide sequence has the following advantages: 1) The modified ATR001 short peptide antigen sequence is more efficient at coating the detection plate, which improves the sensitivity and specificity of anti-ATR001 short peptide antibody detection.

[0021] 2) The modified ATR001 short peptide antigen sequence can be coupled with the carrier bovine serum albumin or other carrier proteins, and used to detect the sample after coating the detection plate. This increases the stability of the coated plate and can significantly improve the sensitivity and specificity of the anti-ATR001 short peptide antibody detection.

[0022] 3) This invention provides the first mature quantitative detection kit for anti-ATR001 short peptide antibodies, providing a detection method for anti-ATR001 short peptide antibodies for the clinical application of ATRQβ-001 therapeutic antihypertensive vaccine, and is used to evaluate the immunogenicity and immune response effect of ATRQβ-001 vaccine. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0024] Example 1 A method for preparing a humanized monoclonal antibody against the short peptide ATR001 includes the following steps: 1) The ATR001 short peptide antigen is conjugated with the carrier protein KLH as an immunoconjugated antigen; wherein, the amino acid sequence of the ATR001 short peptide antigen is shown in SEQ ID No.1: CGGAFHYESQ; The aforementioned carrier protein KLH is Keyhole Limpet Hemocyanin (KLH), a natural high-molecular-weight protein with a molecular weight of 4*10. 5 ~1*10 7 Da. KLH contains two copper ions directly linked to a polypeptide chain. Similar to iron-containing hemoglobin, it readily binds to and dissociates from oxygen, making it a known copper protein that can reversibly bind to oxygen. It appears bluish-green upon oxidation and white upon reduction. One advantage of KLH is that it does not act as a blocking agent and does not interfere with ELISA or Western blotting experiments.

[0025] 2) Mice were immunized with a mixture of immune-conjugated antigen and Freund's adjuvant. Then, spleen cells from the immunized mice were fused with myeloma cells SP2 / 0 and screened to obtain hybridoma cells. 3) Sequencing the obtained hybridoma cells yielded the amino acid sequences of the heavy and light chains of the variable region of the murine monoclonal antibody. The genes of the light and heavy chain variable regions of the murine monoclonal antibody were inserted into an expression vector containing the constant region of the human antibody. The vector was then transformed into mammalian cells to express the chimeric antibody. In this way, the variable regions of the light and heavy chains of the expressed antibody molecule are murine, while the constant regions are human, resulting in a humanized monoclonal antibody against the ATR001 short peptide antigen with the extracellular second loop 181-187 antigenic epitope of angiotensin II receptor type 1.

[0026] The aforementioned humanized anti-ATR001 short peptide antigen monoclonal antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID No. 2: EVQLQQSGPELVKPGTSVKISCKTS GYTFSEYTMH WVKQSHGKSLEWIG GIDPRNGGTTYTPKFKG KATLTVDRSSSTAYMELRRSLTSEDSAVYYCVR YDYRIYYGMDY WGQGTSVTVSS; The amino acid sequence of the light chain is shown in SEQ ID No. 3: QAVVTQESALTTSPGETVTLTC RSSTGPVTTSNAYAN WVQEKPDHLFTGLIG GANIRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHFI FGSGTKVTVL; The heavy chain comprises three variable regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; among them, The amino acid sequence of the heavy chain CDR1 is GYTFSEYTMH, as shown in SEQ ID No. 4; The amino acid sequence of the heavy chain CDR2 is GIDPRNGGTTYTPKFKG, as shown in SEQ ID No. 5; The amino acid sequence of the heavy chain CDR3 is YDYRIYYGMDY, as shown in SEQ ID No. 6; The light chain comprises three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; among them, The amino acid sequence of the light chain CDR1 is RSSTGPVTTSNYAN, as shown in SEQ ID No. 7; The amino acid sequence of the light chain CDR2 is GANIRAP, as shown in SEQ ID No. 8; The amino acid sequence of the light chain CDR3 is ALWYSNHFI, as shown in SEQ ID No. 9.

[0027] Example 2: Preparation of Quantitative Detection Kit I. Preparation of coating antigen: When the ATR001 short peptide antigen is conjugated with bovine serum albumin (BSA), the coated plate becomes a solid-phase ATR001 short peptide antigen-BSA complex. The amino acid sequence of the ATR001 short peptide antigen is shown in SEQ ID No. 1: CGGAFHYESQ; the conjugation steps are as follows: 1. Weigh 2 mg of Sulfo-SMCC reagent, dissolve it completely in 200 μL of pure water, add it to 4 mg of BSA (10 mg / mL), mix well, and incubate in a 37°C water bath for 30 minutes; 2. Add activated BSA to a 10K TFF concentration column, add 50mM PBS (containing 1mM EDTA, pH 7.2), centrifuge at 7500rpm for 15min to remove free Sulfo-SMCC; the TFF concentration column was provided by Millipore Corporation, USA. 3. Weigh 2 mg of the antigen analog peptide, dissolve it completely in 1 mL of 50 mM PBS, add it to activated BSA, and react at room temperature for 2 hours, gently shaking once every 30 minutes. 4. Prepare 1L of 100mM PBS (containing 1mM EDTA, pH 7.2), add the vaccine mixture to a 3.5kDa dialysis bag and dialyze for 24 hours to remove free unreacted peptides; 5. After dialysis, take out the sample for quantitative analysis and store it in aliquots at -80℃, or use it directly for coating.

[0028] II. Antigen Coating: After coupling the ATR001 short peptide antigen fragment with the carrier (bovine serum albumin BSA), it was dissolved in a container containing carbonate buffer at pH 9.6. 100 μl of carbonate buffer per well was used for coating with the corresponding peptide antigen at a concentration of 1.5 μg. After incubation at 4°C for 18 hours, the mixture was washed three times with washing buffer for 1 minute each time. After patting dry, 200 μl of phosphate buffer containing 1% bovine serum albumin was used as blocking buffer per well. The mixture was incubated at 2-8°C for 24 hours to eliminate non-specific reactions. After patting dry, the mixture was stored at 4°C for later use.

[0029] III. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay) (I) Table 1. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay) (Part 1) a. Reagent kit storage conditions and shelf life The kit should be stored at 2–8°C and has a shelf life of 12 months. Once opened, the kit should be stored at 2–8°C for no more than one month. Unused microplate strips should be sealed together with the desiccant in a resealable bag.

[0030] Transport in sealed foam boxes with ice packs, with a transport time not exceeding one week and a transport temperature not exceeding room temperature.

[0031] b. Applicable instruments ELISA reader (including 450nm and 600~650nm wavelength range) V. Kit Testing Procedure: 1. Sample Requirements 1) The sample used in this reagent is human serum.

[0032] 2) Samples containing sodium azide cannot be detected because sodium azide inhibits the activity of horseradish peroxidase; samples containing suspended fibrin or aggregates, or those with severe hemolysis cannot be detected.

[0033] 3) The sample should be free of microorganisms. Aseptically isolated samples can be stored at 2–8°C for 1 week or at temperatures below -18°C for 3 weeks, avoiding repeated freeze-thaw cycles.

[0034] 4) Before use, please place the sample at room temperature (10-30℃) for at least 30 minutes to equilibrate. Frozen samples need to be thawed to room temperature (10-30℃) and mixed well before the experiment.

[0035] 2. Test methods 1) Solution preparation: Dilute one bottle of concentrated washing solution to 1L with distilled or deionized water; 2) Numbering: Number the microwells corresponding to the samples sequentially. Set up 8 wells for standard control on each plate, with concentrations of 200, 150, 100, 50, 25, 12.5, 6.25, and 0 ng / mL, respectively. 3) Sample addition: Dilute the standard to concentrations of 200, 150, 100, 50, 25, 12.5, 6.25, and 0 ng / mL with sample diluent. Dilute the test sample with sample diluent in the range of 1:10 to 1:10000, and add 100 μL to the corresponding well. Gently shake to mix. 4) Incubation: After sealing the plate with sealing film, incubate it in a 37℃ incubator for 60 minutes; 5) Washing: Carefully peel off the sealing film and wash the plate 5 times with a plate washer (or wash it by hand). Soak for 30 seconds each time. Fill each well with washing solution during washing. After the last wash, pat the plate dry on absorbent paper as much as possible. 6) Add enzyme: After shaking well, add 100 μL of enzyme conjugate to each well and gently shake to mix; 7) Incubation: After sealing the plate with another sheet of sealing film, place it in a 37°C incubator for 45 minutes; 8) Washing: Same as step 5.

[0036] 9) Color development: Add 100 μL of color developer to each well and develop the color at 37±1℃ in the dark for 8±1 minutes; 10) Measurement: Add 100 μL of stop solution to each well to stop the reaction, gently shake to mix, and measure the absorbance A value of each well at a wavelength of 450 nm-630 nm within 5 minutes. 11) Result Interpretation: Using the concentration of the standard as x and the corresponding OD450 nm-630 nm value of the standard as y, perform a fourth-order polynomial fitting: Y = a + bX + cX^2 + dX^3 + eX^4. In the formula, Y is the absorbance value A corresponding to the standard, X is the concentration of the standard (ng / mL), and a, b, c, d, and e are all constants. Substitute the absorbance value of the sample well into the fitted equation to calculate the concentration x of the anti-ATR001 short peptide antibody in the sample well; calculate the concentration of the anti-ATR001 short peptide antibody in the sample to be tested based on the following formula; y = x * N; In the formula, y is the concentration of anti-ATR001 short peptide antibody in the sample to be tested, in ng / mL; x represents the concentration of anti-ATR001 short peptide antibody in the well of the coated plate, in ng / mL; N is the dilution factor.

[0037] Example 3 Determination of the standard curve and quantitative range of the above reagent kit The accuracy and coefficient of variation of three standard curves were verified using the above kits: 256, 128, 64, 32, 16, 8, 4, 0 ng / mL; 250, 200, 150, 75, 25, 12.5, 6.25, 0 ng / mL; and 200, 150, 100, 50, 25, 12.5, 6.25, 0 ng / mL. The results showed that the accuracy of 200, 150, 100, 50, 25, 12.5, 6.25, 0 ng / mL was closer to 100%. Furthermore, according to the 2015 edition of the Chinese Pharmacopoeia, section 9012... According to the "Guideline for Validation of Quantitative Analysis Methods for Biological Samples", the mean inter-batch accuracy should generally be within ±15% of the labeled value of the quality control sample, and the coefficient of variation should generally not exceed 15%. The accuracy of the lower limit of quantitation should be within ±20% of the labeled value, and the coefficient of variation of the lower limit of quantitation should not exceed 20%. The quantitation range of the kit is 6.25-200 ng / mL (see Table 2).

[0038] Table 2 Selection and Comparison of Standard Curve Concentration Example 4 Specificity validation of reagent kit detection method Negative samples diluted 1:40, rat serum samples diluted 1:40, and mouse serum samples diluted 1:40, along with PBST washing buffer, 2% BSA-PBST, 1% BSA, and coating buffer, were selected as blank matrices for the quantitative detection of ATR001 short peptide receptor antibodies. According to the 2015 edition of the Chinese Pharmacopoeia, "9012 Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples," an acceptable standard is a response rate lower than 20% of the analyte quantitation limit and lower than 5% of the negative sample response rate.

[0039] Table 3 shows that the concentrations of PBST washing buffer, 2% BSA-PBST, 1% BSA-PBST, coating buffer, 1:40 diluted rat serum sample, and 1:40 diluted mouse serum sample were all below 0. This indicates that the kit can distinguish between the anti-ATR001 short peptide antibody and the internal standard, as well as endogenous components of the matrix or other components in the sample.

[0040] Table 3 Specificity Verification Example 5 Accuracy and precision verification of the reagent kit detection method As needed, select quality control sample concentrations of 200 ng / mL (upper limit of quantitation), 153.6 ng / mL (high concentration), 76.8 ng / mL (medium concentration), 9.6 ng / mL (low concentration), and 6.25 ng / mL (lower limit of quantitation), with 5 replicates, and calculate the concentration, intra-batch accuracy, and intra-batch precision. Perform tests at different times or by different personnel, and calculate the concentration, inter-batch accuracy, and inter-batch precision. According to the 2015 edition of the Chinese Pharmacopoeia, "9012 Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples," the acceptable standards are: the mean accuracy should generally be within ±15% of the labeled value of the quality control sample; for the lower limit of quantitation, it should be within ±20% of the labeled value; the coefficient of variation should generally not exceed 15%, and the coefficient of variation for the lower limit of quantitation should not exceed 20%.

[0041] Table 4 shows that the intra-batch accuracy of the quality control samples was within ±15% of the labeled value, and the intra-batch accuracy of the lower limit of quantitation (LQ) was within ±10% of the labeled value. The intra-batch coefficients of variation for both the quality control samples and the LQ did not exceed 10%. The inter-batch accuracy of the quality control samples was within ±10% of the labeled value, and the inter-batch accuracy of the LQ was within ±15% of the labeled value; the inter-batch coefficients of variation for both the quality control samples and the LQ did not exceed 15%, meeting the acceptable standards.

[0042] Table 4 Accuracy and Precision Verification Example 6 Dilution reliability verification of the reagent kit detection method As needed, quality control samples should be diluted 10-fold, 20-fold, 40-fold, 50-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10000-fold, and 20000-fold, with 5 replicates for each dilution. The concentration, accuracy, and precision should be calculated. According to the 2015 edition of the Chinese Pharmacopoeia, "9012 Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples," the acceptable standards are: accuracy and precision should be within ±15%, and the reliability of the dilutions should cover the dilution factors used for the test samples.

[0043] As shown in Table 5, after low-fold dilution (10x, 20x, 40x, 50x), the accuracy of each concentration of the quality control samples was within ±15% of the labeled value, and the coefficient of variation did not exceed 15%. After high-fold dilution (500x, 1000x, 2000x, 5000x, 10000x, 20000x), the accuracy of each concentration was within ±15% of the labeled value, and the coefficient of variation did not exceed 5%. This indicates that diluting the samples 10x to 20000x does not affect the accuracy and precision of the kit.

[0044] Table 5 Dilution Reliability Verification Example 7 Dilution reliability verification of the reagent kit detection method As needed, incubate the kit components, including PBST washing buffer, 2% BSA-PBST, chromogenic solution, and stop solution, at 37°C or room temperature for 2 hours before detection. Use 10 ng / mL and 192 ng / mL samples for low and high concentration quality control, respectively, with 5 replicates per concentration. Calculate the concentration and accuracy. According to the 2015 edition of the Chinese Pharmacopoeia, "9012 Validation Guidelines for Quantitative Analysis Methods for Biological Samples," the acceptable standard is: when comparing the measured concentration with the labeled concentration, the deviation of the mean of each concentration from the labeled concentration should be within ±15%.

[0045] Table 6 shows that when using reagents treated at 37℃ or room temperature for 2 hours, the accuracy of the quality control samples was within ±15% of the labeled value. This indicates that small changes in the measurement conditions do not affect the results.

[0046] Table 6. Reagent Stability Verification Example 8 Repeatability validation of the reagent kit detection method Thirty-eight serum samples (1:10 dilution) from hypertensive patients that produced AT1R-ECL2 165–191 were selected for quantitative testing three times (at different individuals or at different times) to determine the serum anti-ATR001 short peptide antibody content. As can be seen from Table 7, the average coefficient of variation of the 38 samples is less than 15%, which meets the acceptable standard.

[0047] Table 7 Repeatability Validation All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A humanized monoclonal antibody against the ATR001 short peptide antigen, characterized in that: The monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain includes three variable regions, namely heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and its amino acid sequence is as follows: The heavy chain CDR1 sequence is GYTFSEYTMH. The heavy chain CDR2 sequence is GIDPRNGGTTYTPKFKG. The heavy chain CDR3 sequence is YDYRIYYGMDY; The light chain comprises three variable regions: CDR1, CDR2, and CDR3, with the following amino acid sequences: The light chain CDR1 sequence is RSSTGPVTTSNYAN. The light chain CDR2 sequence is GANIRAP. The light chain CDR3 sequence is ALWYSNHFI.

2. The monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain is as follows: EVQLQQSGPELVKPGTSVKISCKTSGYTFSEYTMHWVKQSHGKSLEWIGGIDPRNGGTTYTPKFKGKATLTVDRSSSTAYMELRSLTSEDSAVYYCVRYDYRIYYGMDYWGQGTSVTVSS; The amino acid sequence of the light chain is as follows: QAVVTQESALTTSPGETVTLTCRSSTGPVTTSNYANWVQEKPDHLFTGLIGGANIRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHFIFGSGTKVTVL.

3. The application of a humanized monoclonal antibody against ATR001 short peptide antigen in the preparation of a detection kit for detecting the content of anti-ATR001 short peptide antibodies produced after subcutaneous injection of ATRQβ-001 therapeutic antihypertensive vaccine in humans.

4. A quantitative detection kit for serum anti-ATR001 short peptide antibody, characterized in that: The detection kit includes a coated plate coated with ATR001 short peptide antigen and a standard; the standard contains the monoclonal antibody as described in claim 1 or 2.

5. The detection kit according to claim 4, characterized in that: The amino acid sequence of the ATR001 short peptide antigen is shown in SEQ ID No. 1: CGGAFHYESQ.

6. The detection kit according to claim 4 or 5, characterized in that: The coated plate is formed by coupling the ATR001 short peptide antigen with the carrier protein bovine serum albumin and then coating it.

7. The detection kit according to claim 4, characterized in that: The standard contained 640 ng / mL of monoclonal antibody.

8. The detection kit according to claim 4, 5, or 7, characterized in that: The test kit includes sample diluent, horseradish peroxidase-labeled goat anti-human IgG antibody, TMB chromogenic solution, stop solution, and 20× concentrated wash solution.

9. The detection kit according to claim 8, characterized in that: The sample diluent is a phosphate buffer containing bovine serum albumin; and the mass fraction of bovine serum albumin in the phosphate buffer is 1%.

10. A method for detecting the concentration of serum anti-ATR001 short peptide antibody using the kit described in claim 4, characterized in that: Includes the following steps: 1) Solution preparation: Dilute the 20× concentrated washing solution to 1× washing solution with distilled or deionized water; 2) Prepare a gradient concentration of standards: Dilute the standards with sample diluent to concentrations of 200, 150, 100, 50, 25, 12.5, 6.25, and 0 ng / mL, respectively; 3) Sample addition: Dilute the sample to be tested with sample diluent, with a dilution ratio ranging from 1:10 to 1:10000; add the diluted standards of different concentrations and the sample to be tested to the corresponding wells and gently shake to mix. 4) Incubation: After sealing the plate with sealing film, place it in an incubator for incubation; 5) Washing: Carefully peel off the sealing film, wash multiple times with a plate washer, soaking each time, and fill each well with washing solution during each wash. On the last wash, pat dry as much as possible on absorbent paper. 6) Add enzyme: After shaking well, add horseradish peroxidase-labeled goat anti-human IgG antibody to each well and gently shake to mix. 7) Incubation: After sealing the plate with another sheet of film, place it in an incubator for incubation; 8) Washing: Same as step 5; 9) Color development: Add TMB developer to each well first, and develop the color in the dark; 10) Measurement: Add stop solution to each well to stop the reaction, gently shake to mix, and measure the absorbance A value of each well at wavelengths of 450 nm-630 nm. 11) Result Interpretation: Using the concentration of the standard as the X value, the corresponding OD value of the standard is... 450 nm-630nm Given the value of y, perform a fourth-order polynomial fitting: Y = a + bX + cX^2 + dX^3 + eX^4 In the formula, Y is the absorbance value A corresponding to the standard, X is the concentration of the standard (ng / mL), and a, b, c, d, and e are all constants. Substitute the absorbance value of the sample well into the fitted equation to calculate the concentration x of the anti-ATR001 short peptide antibody in the sample well; calculate the concentration of the anti-ATR001 short peptide antibody in the sample to be tested based on the following formula; y = x * N; In the formula, y is the concentration of anti-ATR001 short peptide antibody in the sample to be tested, in ng / mL; x represents the concentration of anti-ATR001 short peptide antibody in the well of the coated plate, in ng / mL; N is the dilution factor.

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