Humanized monoclonal antibody of beta1-adrenergic receptor as well as kit and application of humanized monoclonal antibody

Abstract

The invention discloses a humanized monoclonal antibody of a beta1-adrenergic receptor as well as a kit and application of the humanized monoclonal antibody. The amino acid sequence of the humanized monoclonal antibody of the beta1-adrenergic receptor is as shown in SEQ ID No.1. The invention further discloses a preparation method of the humanized monoclonal antibody of the beta1-adrenergic receptor. According to the distribution condition of myocardial antigenic determinants, polypeptides of natural antigenic determinants are designed and synthesized to immunize animals to prepare monoclonal antibodies and carry out humanized expression, so that the antibody detection kit is prepared. The kit can be used for auxiliary diagnosis of autoimmune response activation and prognosis judgment of patients with viral myocarditis and dilated cardiomyopathy, and auxiliary establishment of a patient treatment scheme.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic reagents, specifically to a humanized monoclonal antibody against a β1-adrenergic receptor, its kit, and its applications. Background Technology

[0002] Anticardiomyocyte antibodies (AHAs) are a collective term for antibodies produced by the body against its own myocardial protein molecules, commonly seen in viral myocarditis (VMC) and dilated cardiomyopathy (DCM). Viral infection and its subsequent abnormal immune response are important pathogenic mechanisms of VMC and DCM. Viral infection leads to myocardial damage, exposing hidden myocardial protein antigens, triggering an immune response, and producing AHAs against these self-antigens. According to molecular mimicry theory, myocardial protein antigens and viral proteins have highly homologous sequences. Viral infection of the human body can produce both antiviral antibodies and AHAs against myocardial-specific antigens (Yuan J, et al. The Journal of Immunology 187.6(2011):3451-3452.).

[0003] Anti-β1-adrenergic receptor antibodies act on myocardial β1-adrenergic receptors to produce chronic sympathetic stimulation, which is an important cause of inducing arrhythmias, apoptosis and myocardial remodeling; they can also activate receptor-gated calcium ion channels, leading to calcium overload and damage to cardiomyocytes (Nussinovitch Udi, et al. Clinical Reviews in Allergy and Immunology 44.1(2013):75-83.).

[0004] Foreign literature reports that the detection rate of anti-β1-adrenergic receptor antibodies in DCM patients is 26%-95%, and in VMC patients it is 37%-96%. Clinical studies have relatively small sample sizes, and the low detection rate of these antibodies in healthy individuals is consistent with that in my country. Anti-β1-adrenergic receptor antibodies (AHAs) can predict the risk of death in DCM patients. Stork et al. followed 65 DCM patients for 10.7 years, and the detection rate of anti-β1-AR antibodies was 26%. The all-cause mortality rate was 65% for DCM antibody-positive patients and 44% for antibody-negative patients, showing a significant correlation between DCM antibody positivity and all-cause mortality (Stork, Stefan, et al. Digest of the World Core Medical Journals 152.4(2006):697-704.). Iwata et al. followed up 104 patients with DCM (31±15) for 3 months. The incidence of multifocal premature ventricular contractions and ventricular tachycardia was significantly higher in patients with positive anti-β1-AR antibodies (40 cases) than in those with negative antibodies (90%: 67%, P<0.01; 65%: 34%, P<0.01), and the risk of sudden death was also significantly higher (17.5%: 4.7%, P<0.05) (Iwata, M. Journal of the American College of Cardiology 37 (2001).).

[0005] The mechanism of action of anti-β1-AR is related to calcium channels or β1-AR. Clinically, calcium channel blockers and / or β1-receptor blockers can be added for targeted treatment in the early stages of VCM and DCM. According to AHA test results, early treatment with diltiazem and metoprolol in DCM can effectively inhibit AHA-mediated myocardial injury, improve patient prognosis, and has good safety.

[0006] Natural myocardial antigens are difficult to isolate and purify, and are difficult to preserve for a long time, which limits the clinical application of myocardial antibody detection. Based on the amino acid sequence and antigenic determinant distribution of myocardial autoantibodies, peptides of natural antigenic determinants are designed and synthesized, and these peptides are used as antigens to detect anti-myocardial antibodies in patients. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a humanized monoclonal antibody against β1-adrenergic receptors, its kit, and its applications. Based on the distribution of myocardial antigenic determinants, this invention designs and synthesizes polypeptides of natural antigenic determinants for immunization of animals to prepare monoclonal antibodies, which are then humanized and expressed to obtain humanized monoclonal antibodies against β1-adrenergic receptors. An antibody detection kit is then prepared. This kit can be used to assist in the diagnosis of autoimmune activation and prognosis assessment in patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM), and to aid in the establishment of treatment plans.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0009] This invention provides a humanized monoclonal antibody against a β1-adrenergic receptor, the humanized monoclonal antibody comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID No. 1:

[0010] EVQLQQPGAELVRPGASLKLSCKASGYTFTNYWMNWVK QRPGQGLEWIGIIDPSDSKTHYNQMFKDKATLTVDKSSTTAYMQ LRSLTSEDSAVYYCATGGFRYWGQGTLVTVSS;

[0011] The amino acid sequence of the light chain is shown in SEQ ID No. 2:

[0012] DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWL LQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAE DLGVYYCWQGTHFPQTFGGGTKLEIN.

[0013] Furthermore, the heavy chain includes three variable regions, namely heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; wherein,

[0014] The amino acid sequence of heavy chain CDR1 is NYWMN.

[0015] The amino acid sequence of the heavy chain CDR2 is IIDPSDSKTHYNQMFKD.

[0016] The amino acid sequence of the heavy chain CDR3 is GGFRY;

[0017] The light chain includes three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; wherein,

[0018] The amino acid sequence of the light chain CDR1 is KSSQSLLDSDGKTYLN.

[0019] The amino acid sequence of the light chain CDR2 is LVSKLDS.

[0020] The amino acid sequence of the light chain CDR3 is WQGTHFPQT.

[0021] The present invention also provides the application of the above-mentioned humanized monoclonal antibody in the preparation of an autoantibody detection kit.

[0022] Furthermore, the test kit is

[0023] (1) Enzyme-linked immunosorbent assay (ELISA) kit;

[0024] (2) Colloidal gold test strip detection kit;

[0025] (3) Chemiluminescence detection kit;

[0026] (4) Flow cytometer detection kit.

[0027] The present invention also provides a serum anti-β1-adrenergic receptor autoantibody detection kit, the detection kit comprising a standard and a coated plate coated with the above-mentioned β1-adrenergic receptor antigen analog polypeptide, wherein the standard contains a standard solution of the above-mentioned humanized monoclonal antibody.

[0028] Furthermore, the standard contains 1280 ng / mL of humanized monoclonal antibody against β1-adrenergic receptor.

[0029] Furthermore, the amino acid sequence of the β1-adrenergic receptor antigen is shown in SEQ ID No. 3:

[0030] WRAESDEARRCYNDPK.

[0031] Furthermore, the enzyme-linked immunosorbent assay (ELISA) kit also includes a sample diluent, an enzyme conjugate (horseradish peroxidase-labeled goat anti-human IgG antibody), a TMB chromogenic solution, a stop solution, and a concentrated washing solution. The sample diluent is prepared by dissolving 1 g of BSA in 100 mL of 0.1 M phosphate buffer at pH 7.4.

[0032] Furthermore, the coating plate containing the β1-adrenergic receptor antigen analog polypeptide is prepared by the following steps:

[0033] 1) Activation: First, dissolve 1 mg of BSA (bovine serum albumin) in filtered PBS, then dissolve 2 mg of Sulfo-SMCC (sodium salt of 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester) in DMSO (dimethyl sulfoxide). Adjust the SMCC concentration to 20 mg / mL with filtered PBS. After mixing the two, place them in a 37°C water bath and incubate for 30 min, shaking once every 10 min.

[0034] 2) Removal of free SMCC: Centrifuge the solution after mixing and reaction in step 1) using a 10 kDa TFF ultrafiltration tube to obtain activated BSA;

[0035] 3) Conjugation: Dissolve 1 mg of β1-adrenergic receptor antigen analog peptide in filtered PBS, then mix with the activated BSA from step 2), and place on a shaker at room temperature for 2 h to obtain the conjugate.

[0036] 4) Removal of free short peptides: Place the conjugate obtained in step 3) in a 3.5 kDa dialysis bag and dialyze with 0.01 mol / L PBS for 24 h, changing the medium every 8 h during the process, and collect the conjugate after dialysis.

[0037] 5) Coating: After measuring the concentration of the conjugate after dialysis in step 4), adjust the concentration to 4 μg / mL with carbonate buffer, add 100 μL / well to the microplate, and incubate at 2-8℃ for 18-24 h.

[0038] 6) After washing with washing solution and patting dry, block with phosphate buffer containing 1% bovine serum albumin and 0.625% cysteine. Incubate at 37°C for 2 hours, then pat dry, seal and store in a refrigerator at 2-8°C to obtain the corresponding coated plate for later use.

[0039] This invention also provides a method for detecting serum anti-β1-adrenergic receptor autoantibody levels based on the above-described kit, comprising the following steps:

[0040] 1) Dilute the sample to be tested (serum) and the standard to different gradients and add them to the corresponding wells of the coated plate;

[0041] 2) After sealing the coated board with sealing film, incubate and wash;

[0042] 3) Add the enzyme conjugate to the wells of the coated plate, gently shake to mix; then seal the coated plate with sealing film and incubate, then wash.

[0043] 4) Add color developer and develop color in the dark;

[0044] 5) Stop the reaction with the stop solution, gently shake to mix, and measure the absorbance A value of each well;

[0045] 6) Result Interpretation: Using the concentration of the standard as X and the corresponding absorbance value A as Y, perform a quadratic polynomial fitting: Y = aX 2 +bX+c,

[0046] In the formula, Y is the absorbance value A corresponding to the standard.

[0047] X represents the concentration of the standard (ng / mL);

[0048] a, b, and c are all constants;

[0049] The concentration x of anti-β1-adrenergic receptor antibody in the sample well is then calculated based on the fitted equation; the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested is calculated based on the following formula;

[0050] y = x * N;

[0051] In the formula, y is the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested, (ng / mL);

[0052] x represents the concentration of anti-β1-adrenergic receptor antibody in the well of the coated plate (ng / mL);

[0053] N is the dilution factor.

[0054] The beneficial effects of this invention are:

[0055] This invention provides the first mature quantitative detection kit for anti-β1-adrenergic receptor autoantibodies, which is used to detect anti-β1-adrenergic receptor autoantibodies and assist in the diagnosis of viral myocarditis (VMC) and dilated cardiomyopathy (DCM), and guide the establishment of treatment plans for patients. Detailed Implementation

[0056] 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.

[0057] Example 1

[0058] A method for preparing a humanized monoclonal antibody against β1-adrenergic receptors includes the following steps:

[0059] 1) The β1-adrenergic receptor antigen is coupled to the carrier protein KLH as an immunoconjugated antigen; the amino acid sequence of the β1-adrenergic receptor antigen is shown in SEQ ID No. 3:

[0060] WRAESDEARRCYNDPK;

[0061] 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.

[0062] 3) Sequencing the obtained hybridoma cells yields 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 are inserted into an expression vector containing the constant region of the human antibody. The vector is then transformed into mammalian cells to express chimeric antibodies. In this way, the variable regions of the light and heavy chains of the expressed antibody molecules are murine, while the constant regions are human, resulting in a humanized monoclonal antibody against the β1-adrenergic receptor antigen.

[0063] The aforementioned humanized 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. 1:

[0064] EVQLQQPGAELVRPGASLKLSCKASGYTFT NYWMN WVKQ RPGQGLEWIG IIDPSDSKTHYNQMFKD KATLTVDKSSTTAYMQL RSLTSEDSAVYYCAT GGFRY WGQGTLVTVSS;

[0065] The amino acid sequence of the light chain is shown in SEQ ID No. 2:

[0066] DIVMTQTPLTLSVTIGQPASISC KSSQSLLDSDGKTYLN WLL QRPGQSPKRLIY LVSKLDS GVPDRFTGSGSGTDFTLKISRVEAED LGVYYC WQGTHFPQT FGGGTKLEIN.

[0067] The heavy chain comprises three variable regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; among them,

[0068] The amino acid sequence of heavy chain CDR1 is NYWMN.

[0069] The amino acid sequence of the heavy chain CDR2 is IIDPSDSKTHYNQMFKD.

[0070] The amino acid sequence of the heavy chain CDR3 is GGFRY;

[0071] The light chain comprises three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; among them,

[0072] The amino acid sequence of the light chain CDR1 is KSSQSLLDSDGKTYLN.

[0073] The amino acid sequence of the light chain CDR2 is LVSKLDS.

[0074] The amino acid sequence of the light chain CDR3 is WQGTHFPQT.

[0075] Example 2

[0076] I. Preparation of coating antigen (enzyme-linked immunosorbent assay):

[0077] When β1-adrenergic receptor antigen is conjugated with bovine serum albumin (BSA), the coated plate becomes a solidified β1-adrenergic receptor antigen-BSA complex; the conjugation steps are as follows:

[0078] 1. First, dissolve 1 mg of BSA (bovine serum albumin) in filtered PBS (adjust the concentration to 10 mg / ml). Then, weigh 2 mg of Sulfo-SMCC reagent, dissolve it completely in 20 μL of DMSO, add 80 μL of PBS and mix well. Add this mixture to the dissolved 1 mg of BSA (10 mg / ml), mix well, and incubate in a 37°C water bath for 30 min, shaking once every 10 min.

[0079] 2. Add the activated BSA to a 10K TFF concentration column (purchased from Millipore, USA), add 10mM PBS (pH 7.2), centrifuge at 5,000g, add 10mM PBS (pH 7.2) again, centrifuge at 5,000g to fully remove free Sulfo-SMCC;

[0080] 3. Weigh 1 mg of antigenic peptide, dissolve it thoroughly in 100 μL PBS (10 mM), add it to activated BSA, and shake the mixture on a shaker at room temperature for 2 hours, gently shaking once every 30 minutes.

[0081] 4. Prepare 1L of 10mM PBS (pH 7.2), add the conjugate to a 3.5KD dialysis bag and dialyze for 24 hours, changing the medium every 8 hours to remove free unreacted peptides, and collect the dialyzed conjugate.

[0082] 5. After dialysis, take out the sample for quantitative analysis and store it in aliquots at -80℃, or use it directly for coating.

[0083] II. Antigen Coating:

[0084] 1. After determining the concentration of the conjugate obtained from dialysis in step one, adjust the concentration to 4 μg / mL with carbonate buffer. Coat each well with 100 μl of carbonate buffer containing 0.4 μg of the corresponding polypeptide antigen. Incubate at 4°C for 18 hours, then wash three times with washing buffer for 3 minutes each time.

[0085] 2. After patting dry, use phosphate buffer containing 1% bovine serum albumin and 0.625% cysteine ​​as the blocking solution, 150 μl per well, and incubate at 37°C for 2 h to block and eliminate non-specific reactions. Wash 3 times with washing solution, pat dry, and obtain the immobilized β1-adrenergic receptor antigen coated plate, which is stored at 2-8°C for later use.

[0086] III. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay)

[0087] The serum anti-β1-adrenergic receptor autoantibody detection kit includes a coated plate with β1-adrenergic receptor antigen, standards, sample diluent, enzyme conjugate, TMB chromogenic solution, stop solution, and concentrated wash buffer; the specific components are as follows:

[0088] Table 1. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay) (Part 1)

[0089]

[0090] Note a. Reagent kit storage conditions and expiration date

[0091] 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.

[0092] Transport in sealed foam boxes with ice packs, with a transport time not exceeding one week and a transport temperature not exceeding room temperature.

[0093] b. Applicable instruments

[0094] ELISA reader (including 450nm and 600-650nm wavelength range)

[0095] IV. Determination of the standard curve and quantitative range of the above (enzyme-linked immunosorbent assay) kit

[0096] The accuracy and coefficient of variation of the three standard curves (64, 32, 16, 8, 4, 2, 1, 0 ng / mL and 128, 64, 32, 16, 8, 4, 2, 1, 0 ng / mL and 64, 32, 16, 8, 4, 2, 0 ng / mL) were verified using the above (enzyme-linked immunosorbent assay) kit.

[0097] As shown in Table 2: the accuracy of 64, 32, 16, 8, 4, 2, 1, and 0 ng / mL is closer to 100% and the cv% value is lower;

[0098] Furthermore, according to the "Guideline for Validation of Quantitative Analysis Methods for Biological Samples" in the 2015 edition of the Chinese Pharmacopoeia, the mean accuracy between batches should generally be within ±15% of the labeled value of the quality control sample, 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, the coefficient of variation of the lower limit of quantitation should not exceed 20%, and the quantitation range of the kit is 3-50 ng / mL (corresponding to an antibody concentration of 60-1000 ng / mL in serum samples).

[0099] Table 2. Determination of standard curve and quantitative range of the reagent kit

[0100]

[0101] V. Definition of the negative range for serum anti-β1-adrenergic receptor antibody detection using (enzyme-linked immunosorbent assay) kit

[0102] Forty-one negative serum samples of anti-β1-adrenergic receptor antibody were selected as needed for quantitative detection of anti-β1-adrenergic receptor antibody. The content of anti-β1-adrenergic receptor antibody in serum was measured, and 2.1 times the absorbance value A of negative serum was used as the positive cutoff point to calculate the negative range of anti-β1-adrenergic receptor antibody in the kit.

[0103] As shown in Table 3, the negative range of the anti-β1-adrenergic receptor antibody in the kit is 0-172.722 ng / mL.

[0104] Table 3. Detection values ​​of anti-β1-adrenergic receptor antibody in negative samples

[0105]

[0106]

[0107] VI. (Enzyme-Linked Immunosorbent Assay) Kit Detection Procedure:

[0108] 1. Sample Requirements

[0109] 1) The sample used in this reagent is human serum.

[0110] 2) Samples containing sodium azide cannot be tested because sodium azide inhibits the activity of horseradish peroxidase; samples containing suspended fibrin or aggregates, or those with severe hemolysis cannot be tested.

[0111] 3) The sample should be free of microorganisms. Aseptically isolated samples can be stored at 2–8°C for 1 week or at -18°C for 3 weeks, avoiding repeated freeze-thaw cycles.

[0112] 4) Before use, please place the sample at room temperature (10-30℃) for more than 30 minutes to equilibrate. Frozen samples need to be thawed to room temperature (10-30℃) and mixed before the experiment.

[0113] 2. Test methods

[0114] 1) Solution preparation: Dilute one bottle of concentrated washing solution to 1L with distilled or deionized water;

[0115] 2) Numbering: Number the microwells corresponding to the samples in sequence. Set up 8 wells for standard control on each plate with concentrations of 64, 32, 16, 8, 4, 2, 1, and 0 ng / mL, respectively.

[0116] 3) Sample addition: Dilute the standard to a concentration of 64, 32, 16, 8, 4, 2, 1, or 0 ng / mL with sample diluent, and dilute the test sample with sample diluent at a dilution gradient of 1:20. Add the sample to the corresponding well (100 μL per well) and gently shake to mix.

[0117] 4) Incubation: After sealing the plate with sealing film, incubate it in a 37℃ incubator for 60 minutes;

[0118] 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.

[0119] 6) Add enzyme: After shaking well, add 100 μL of enzyme conjugate to each well and gently shake to mix.

[0120] 7) Incubation: After sealing the plate with another sheet of sealing film, place it in a 37℃ incubator for 45 minutes;

[0121] 8) Washing: Same as step 5.

[0122] 9) Color development: Add 100 μL of color developer to each well and develop the color at 37±1℃ in the dark for 10±1 min;

[0123] 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 within 5 min.

[0124] 11) Result Interpretation: Using the concentration of the standard as X and the corresponding absorbance value A as Y, perform a quadratic polynomial fitting: Y = aX 2 +bX+c,

[0125] In the formula, Y represents the absorbance value A corresponding to the standard.

[0126] X represents the concentration of the standard (ng / mL).

[0127] a, b, and c are all constants;

[0128] The concentration x of anti-β1-adrenergic receptor antibody in the well of the coated plate is then calculated based on the fitted equation; the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested is calculated based on the following formula.

[0129] y = x * N;

[0130] In the formula, y is the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested, (ng / mL);

[0131] x represents the concentration of anti-β1-adrenergic receptor antibody within the wells of the coated plate.

[0132] (ng / mL);

[0133] N is the dilution factor, N = 20.

[0134] Example 4

[0135] The above (enzyme-linked immunosorbent assay) kit detects positive samples of anti-β1-adrenergic receptor antibodies.

[0136] Sixty-nine serum samples that were positive for anti-β1-adrenergic receptor antibodies were selected as needed for quantitative detection of anti-β1-adrenergic receptor antibodies to determine the serum anti-β1-adrenergic receptor antibody content.

[0137] The data in Table 4 show that the average coefficient of variation for the 69 samples was 8.57%, and the percentage of samples with a coefficient of variation below 15% was 85.51% (59 / 69), indicating that the kit has good repeatability in multiple tests.

[0138] Table 4. Repeatability of detection in positive samples of anti-β1-adrenergic receptor antibody

[0139]

[0140]

[0141]

[0142]

[0143] Example 5

[0144] The (enzyme-linked immunosorbent assay) kit is used to detect anti-β1-adrenergic receptor antibodies and to aid in the diagnosis of viral myocarditis (VMC) and dilated cardiomyopathy (DCM), assessing its sensitivity and specificity.

[0145] The above-mentioned kit was used to perform confirmatory testing on 39 clinically diagnosed patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM) to verify the detection rate of the above-mentioned diagnostic kit.

[0146] The test results are shown in Table 5: The detection rate of anti-β1-adrenergic receptor antibodies in patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM) using the kit was 92.31% (36 / 39); indicating that the kit has good sensitivity and specificity for patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM); thus, it can assist in the diagnosis of viral myocarditis (VMC) and dilated cardiomyopathy (DCM) and help establish a treatment plan for patients.

[0147] Table 5. Detection results of anti-β1-adrenergic receptor antibodies in patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM).

[0148]

[0149]

[0150] 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 a β1-adrenergic receptor antigen, characterized in that: The humanized 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. 1: EVQLQQPGAELVRPGASLKLSCKASGYTFTNYWMNWVK QRPGQGLEWIGIIDPSDSKTHYNQMFKDKATLTVDKSSTTAYMQ LRSLTSEDSAVYYCATGGFRYWGQGTLVTVSS; The amino acid sequence of the light chain is shown in SEQ ID No. 2: DIVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWL LQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAE DLGVYYCWQGTHFPQTFGGGTKLEIN.

2. The humanized monoclonal antibody according to claim 1, characterized in that: The heavy chain includes three variable regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; wherein... The amino acid sequence of heavy chain CDR1 is NYWMN. The amino acid sequence of the heavy chain CDR2 is IIDPSDSKTHYNQMFKD. The amino acid sequence of the heavy chain CDR3 is GGFRY; The light chain includes three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; wherein, The amino acid sequence of the light chain CDR1 is KSSQSLLDSDGKTYLN. The amino acid sequence of the light chain CDR2 is LVSKLDS. The amino acid sequence of the light chain CDR3 is WQGTHFPQT.

3. The use of the humanized monoclonal antibody according to claim 1 in the preparation of a detection kit.

4. The application according to claim 3, characterized in that: The test kit is (1) Enzyme-linked immunosorbent assay (ELISA) kit; (2) Colloidal gold test strip detection kit; (3) Chemiluminescence detection kit; (4) Flow cytometer detection kit.

5. An enzyme-linked immunosorbent assay (ELISA) kit, characterized in that, The test kit also includes standards and a coated plate coated with β1-adrenergic receptor antigen, wherein the standards contain a standard solution of the humanized monoclonal antibody as described in claim 1.

6. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, characterized in that: The standard contained 1280 ng / mL of humanized monoclonal antibody.

7. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, characterized in that: The amino acid sequence of the β1-adrenergic receptor antigen is shown in SEQ ID No. 3: WRAESDEARRCYNDPK.

8. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, 6, or 7, characterized in that: The autoantibody detection kit also includes sample diluent, enzyme conjugate, TMB chromogenic solution, stop solution, and concentrated washing solution, wherein... The sample diluent was prepared by dissolving 1 g of BSA in 100 mL of 0.1 M phosphate buffer at pH 7.

4.

9. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5 or 7, characterized in that: The coated plate containing the β1-adrenergic receptor antigen was prepared by the following steps: 1) First, dissolve 1 mg BSA in filtered PBS, then dissolve 2 mg Sulfo-SMCC in DMSO. Adjust the Sulfo-SMCC concentration to 20 mg / mL with filtered PBS. After mixing the two, place them in a 37°C water bath and incubate for 30 min. Gently shake once every 10 min. 2) Centrifuge the solution after the reaction in step 1) using a 10 kDa TFF ultrafiltration tube to obtain activated BSA; 3) Dissolve 1 mg of β1-adrenergic receptor antigen in filtered PBS, then mix it with the activated BSA from step 2), and place it on a shaker at room temperature for 2 hours, gently shaking it once every 30 minutes to obtain the conjugate. 4) Place the conjugate obtained in step 3) in a 3.5 kDa dialysis bag and dialyze with 0.01 mol / L PBS for 24 h, changing the solution every 8 h during the process, and collect the conjugate after dialysis. 5) Coating: After measuring the concentration of the conjugate after dialysis in step 4), adjust the concentration to 4 μg / mL with carbonate buffer, add 100 μL / well to the microplate, and incubate at 2-8℃ for 18-24 h. 6) After washing with washing solution and patting dry, block with phosphate buffer containing 1% BSA and 0.625% cysteine ​​as blocking solution. Incubate at 37°C for 2 hours, then pat dry, seal and store in a refrigerator at 2-8°C to obtain the corresponding coated plate for later use.

10. A method for detecting serum anti-β1-adrenergic receptor autoantibody content based on the enzyme-linked immunosorbent assay kit according to claim 5, characterized in that: Includes the following steps: 1) Dilute the test samples and standards to different gradients and add them to the wells of the corresponding coated plates; 2) After sealing the coated board with sealing film, incubate and wash; 3) Add the enzyme conjugate to the wells of the coated plate and gently shake to mix. Then cover the coated board with sealing film and incubate it, then wash it; 4) Add color developer and develop color in the dark; 5) Stop the reaction with the stop solution, gently shake to mix, and measure the absorbance A value of each well; 6) Result Interpretation: Using the concentration of the standard as X and the corresponding absorbance value A as Y, perform a quadratic polynomial fitting: Y = aX 2 +bX+c, In the formula, Y represents the absorbance value A corresponding to the standard. X represents the concentration of the standard, in ng / mL; a, b, and c are all constants; The concentration x of anti-β1-adrenergic receptor antibody in the sample well is then calculated based on the fitted equation; the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested is calculated based on the following formula; y = x * N; In the formula, y represents the concentration of anti-β1-adrenergic receptor antibody in the sample to be tested, in ng / mL; x represents the concentration of anti-β1-adrenergic receptor antibody in the wells of the coated plate, in ng / mL; N is the dilution factor.

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