L-type calcium channel humanized monoclonal antibody as well as kit and application thereof

Abstract

The invention discloses a humanized monoclonal antibody of an L-type calcium channel as well as a kit and application of the humanized monoclonal antibody, the humanized monoclonal antibody comprises a heavy chain and a light chain, and amino acid sequences of the heavy chain and the light chain are respectively shown as SEQ ID No.1 and SEQ ID No.2; according to the distribution condition of myocardial antigenic determinants, a polypeptide of a natural antigenic determinant, namely an L-type calcium channel antigen, is designed and synthesized, an animal is immunized to prepare a monoclonal antibody, humanized expression is carried out, and the antibody detection kit is prepared from the monoclonal antibody for application. The kit can be used for auxiliary diagnosis of 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 for L-type calcium channels, 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] L-type calcium channels are important ion channels on the myocardial cell membrane. As the main gateway for calcium ions to enter the cell, they play an important role in the generation and conduction of normal cardiac excitability. At the same time, they are also an important mechanism for triggering intracellular calcium release and excitation-contraction coupling (Tu Danna et al. Chinese Journal of Immunology 21.10(2005):4.).

[0004] Anti-L-type calcium channel antibodies act on the α1 subunit of L-type Ca2+ channels in the cardiomyocyte membrane, activating calcium channels and increasing Ca2+ influx, prolonging the effective refractory period of the cardiomyocyte action potential, and causing early afterpolarization, inducing ventricular arrhythmias, and ultimately leading to ventricular tachycardia, which may result in sudden death. Xiao et al. demonstrated that the incidence of sudden death is high in DCM patients with positive anti-L-type calcium channel antibodies, and proposed that anti-L-type calcium channel antibodies can serve as a marker for predicting sudden death in dilated cardiomyopathy. The calcium antagonist diltiazem has a protective effect on the myocardium in early dilated cardiomyopathy (Xiao, Hua, et al. European Journal of Heart Failure Supplements 13.3(2014):264-270.).

[0005] 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. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a humanized monoclonal antibody for L-type calcium channels, its kit, and its application. Based on the distribution of myocardial antigenic determinants, this invention designs and synthesizes polypeptides of natural antigenic determinants, which are L-type calcium channel antigens. Monoclonal antibodies are prepared by immunizing animals and then expressed humanized to obtain humanized monoclonal antibodies. An antibody detection kit is then prepared for application. This kit can be used to assist in the diagnosis of viral myocarditis (VMC) and dilated cardiomyopathy (DCM) and to assist in the establishment of treatment plans for patients.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0008] This invention provides a humanized monoclonal antibody for L-type calcium channels, 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:

[0009] EVQLVESGPGLVKPSQSLSLTCSVTGYSITSAFYWNWIRQFP GNKLEWMGCISYDGSFHLNPSLKNRISVTRDTSKNLFFLRLNSV TSEDTAAYFCTRGTDYWGQGTTLTVSS;

[0010] The amino acid sequence of the light chain is shown in SEQ ID No. 2:

[0011] DIVMTQTPLSLFVSLGDQASISCRSSQSLVHSSGNTYLHWY LQKPGQSPKLLIYKLSSRFSGVPDRFSGSGSGTDFTLKISRVEAE DLGVYFCSQSTYVPWTFGGGTKLEIN.

[0012] Furthermore, the heavy chain includes three variable regions, namely heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; wherein,

[0013] The amino acid sequence of the heavy chain CDR1 is SAFYWN.

[0014] The amino acid sequence of the heavy chain CDR2 is CISYDGSFHLNPSLKN.

[0015] The amino acid sequence of the heavy chain CDR3 is GTDY:

[0016] The light chain includes three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; wherein,

[0017] The amino acid sequence of the light chain CDR1 is RSSQSLVHSSGNTYLH.

[0018] The amino acid sequence of the light chain CDR2 is KLSSRFS.

[0019] The amino acid sequence of the light chain CDR3 is SQSTYVPWT.

[0020] The present invention also provides the application of the above-mentioned humanized monoclonal antibody in the preparation of an autoantibody detection kit.

[0021] Furthermore, the test kit is

[0022] (1) Enzyme-linked immunosorbent assay (ELISA) kit;

[0023] (2) Colloidal gold test strip detection kit;

[0024] (3) Chemiluminescence detection kit;

[0025] (4) Flow cytometer detection kit.

[0026] The present invention also provides an enzyme-linked immunosorbent assay (ELISA) kit, the kit further comprising a standard and a coated plate coated with L-type calcium channel antigen, wherein the standard contains a standard solution of the aforementioned humanized monoclonal antibody.

[0027] Furthermore, the standard contains 1280 ng / mL of humanized monoclonal antibody against L-type calcium channels.

[0028] Furthermore, the amino acid sequence of the L-type calcium channel antigen is shown in SEQ ID No. 3:

[0029] VNENTRMYIPEENHQ.

[0030] Furthermore, the enzyme-linked immunosorbent assay (ELISA) kit also includes sample diluent, enzyme conjugate (horseradish peroxidase-labeled goat anti-human IgG antibody), TMB chromogenic solution, stop solution, and concentrated washing solution, wherein...

[0031] The sample diluent was prepared by dissolving 1 g of BSA in 100 mL of 0.1 M phosphate buffer at pH 7.4.

[0032] Furthermore, the L-type calcium channel antigen-coated plate in the enzyme-linked immunosorbent assay (ELISA) kit 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) 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 L-type calcium channel 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 to obtain the conjugate;

[0036] 4) Removal of free peptides: The conjugate obtained in step 3) was placed in a 3.5 kDa dialysis bag and dialyzed with 0.01 mol / L PBS for 24 h, with the solution changed every 8 h. The conjugate was then collected after dialysis.

[0037] 5) Coating: After measuring the concentration of the conjugate after dialysis in step 4), adjust the concentration to 10 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-L-type calcium channel autoantibodies based on the above-described enzyme-linked immunosorbent assay (ELISA) 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 represents the absorbance value A corresponding to the standard.

[0047] X represents the concentration of the standard, in ng / mL;

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

[0049] The concentration x of anti-L-type calcium channel autoantibody in the sample well is then calculated based on the fitted equation; the concentration of anti-L-type calcium channel autoantibody in the sample to be tested is calculated based on the following formula: y = x * N;

[0050] In the formula, y represents the concentration of anti-L-type calcium channel autoantibodies in the sample to be tested, (ng / mL);

[0051] x represents the concentration of anti-L-type calcium channel autoantibodies in the wells of the coated plate (ng / mL).

[0052] N is the dilution factor.

[0053] The beneficial effects of this invention are:

[0054] This invention provides the first mature quantitative detection kit for anti-L-type calcium channel autoantibodies, which can be used to detect anti-L-type calcium channel 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

[0055] 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.

[0056] Example 1

[0057] The method for preparing humanized monoclonal antibodies against L-type calcium channel antigens includes the following steps:

[0058] 1) L-type calcium channel antigen is coupled to the carrier protein KLH as an immunocoupled antigen; wherein, the amino acid sequence of the L-type calcium channel antigen is shown in SEQ ID No. 3:

[0059] VNENTRMYIPEENHQ;

[0060] 2) Mice were immunized with a mixture of immune-coupled 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.

[0061] 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 L-type calcium channel humanized monoclonal antibodies.

[0062] 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:

[0063] EVQLVESGPGLVKPSQSLSLTCSVTGYSIT SAFYWN WIRQFP GNKLEWMG CISYDGSFHLNPSLKN RISVTRDTSKNLFFLRLNSV TSEDTAAYFCTR GTDY WGQGTTLTVSS; The heavy chain includes three variable regions: heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; among them,

[0064] The amino acid sequence of the heavy chain CDR1 is SAFYWN.

[0065] The amino acid sequence of the heavy chain CDR2 is CISYDGSFHLNPSLKN.

[0066] The amino acid sequence of the heavy chain CDR3 is GTDY;

[0067] The amino acid sequence of the light chain is shown in SEQ ID No. 2:

[0068] DIVMTQTPLSLFVSLGDQASISC RSSQSLVHSSGNTYLH WY LQKPGQSPKLLIY KLSSRFS GVPDRFSGSGSGTDFTLKISRVEAE DLGVYFC SQSTYVPWT FGGGTKLEIN; The light chain includes three variable regions: light chain CDR1, light chain CDR2, and light chain CDR3; among them,

[0069] The amino acid sequence of the light chain CDR1 is RSSQSLVHSSGNTYLH.

[0070] The amino acid sequence of the light chain CDR2 is KLSSRFS.

[0071] The amino acid sequence of the light chain CDR3 is SQSTYVPWT.

[0072] Example 2

[0073] I. Preparation of coating antigen:

[0074] L-type calcium channel antigen was coupled to bovine serum albumin (BSA), and the coated plate was a solid-phase L-type calcium channel antigen-BSA complex. The coupling steps are as follows:

[0075] 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, mix well, and incubate in a 37°C water bath for 30 minutes, shaking once every 10 minutes.

[0076] 2. Add the activated BSA to a 10K TFF concentration column (purchased from Millipore, USA), add 10mM PBS (pH 7.2), centrifuge once at 5,000g, add 10mM PBS (pH 7.2) again, centrifuge at 5,000g to fully remove free Sulfo-SMCC;

[0077] 3. Weigh 1 mg of antigen, 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.

[0078] 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.

[0079] 5. After dialysis, take out the sample for quantitative analysis and store it in aliquots at -80℃, or use it directly for coating.

[0080] II. Antigen Coating:

[0081] 1. After determining the concentration of the conjugate after dialysis in step one, adjust the concentration to 10 μg / mL with carbonate buffer. Coat each well with 100 μl of carbonate buffer containing 1 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.

[0082] 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 nonspecific reactions. Wash 3 times with washing solution, pat dry, and obtain the coated plate of the above-mentioned L-type calcium channel antigen in a solid phase. Store at 2-8°C for later use.

[0083] III. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay)

[0084] The serum anti-L-type calcium channel autoantibody detection kit includes a coated plate containing L-type calcium channel antigen, standards, sample diluent, enzyme conjugate, TMB chromogenic solution, stop solution, and concentrated wash buffer; the specific components are as follows:

[0085] Table 1. Main Components of the Reagent Kit (Enzyme-Linked Immunosorbent Assay) (Part 1)

[0086]

[0087] Note a. Reagent kit storage conditions and expiration date

[0088] 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.

[0089] Transport in sealed foam boxes with ice packs, with a transport time not exceeding one week and a transport temperature not exceeding room temperature.

[0090] b. Applicable instruments

[0091] ELISA reader (including 450nm and 600-650nm wavelength range).

[0092] IV. Determination of the standard curve and quantitative range of the above (enzyme-linked immunosorbent assay) kit

[0093] The accuracy and coefficient of variation of the three standard curves (320, 160, 80, 40, 20, 10, 5, 0 ng / mL and 640, 320, 160, 80, 40, 20, 10, 5, 0 ng / mL and 320, 160, 80, 40, 20, 10, 0 ng / mL) were verified using the above (enzyme-linked immunosorbent assay) kits.

[0094] As shown in Table 2, the accuracy of 320, 160, 80, 40, 20, 10, 5, and 0 ng / mL is closer to 100% and the cv% value is lower. 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 15–512 ng / mL (corresponding to an antibody concentration in serum of 300–10240 ng / mL).

[0095] Table 2. Determination of standard curve and quantitative range of the reagent kit

[0096]

[0097]

[0098] V. Definition of the negative range for the detection of anti-L-type calcium channel antibodies in serum using the (enzyme-linked immunosorbent assay) kit

[0099] Forty-five serum samples that were negative for anti-L-type calcium channel antibodies were selected as needed for quantitative detection of anti-L-type calcium channel antibodies. The content of anti-L-type calcium channel antibodies in the serum was measured, and 2.1 times the absorbance value A of the negative serum was used as the positive cutoff point to calculate the negative range of anti-L-type calcium channel antibodies in the kit.

[0100] As can be seen from Table 3, the negative range of the anti-L-type calcium channel antibody in the kit should be 0 to 470.639 ng / mL.

[0101] Table 3. Detection values ​​of anti-L-type calcium channel antibody in negative samples

[0102]

[0103]

[0104]

[0105] VI. Detection procedure of the above (enzyme-linked immunosorbent assay) kit:

[0106] 1. Sample Requirements

[0107] 1) The sample used in this reagent is human serum.

[0108] 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.

[0109] 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.

[0110] 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 before the experiment.

[0111] 2. Test methods

[0112] 1) Solution preparation: Dilute one bottle of concentrated washing solution to 1L with distilled or deionized water;

[0113] 2) Numbering: Number the microwells corresponding to the samples in sequence. Set up 8 wells for standard control on each plate with concentrations of 320, 160, 80, 40, 20, 10, 5, and 0 ng / mL, respectively.

[0114] 3) Sample addition: Dilute the standard to a concentration of 320, 160, 80, 40, 20, 10, 5, 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.

[0115] 4) Incubation: After sealing the plate with sealing film, incubate it in a 37℃ incubator for 60 minutes;

[0116] 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.

[0117] 6) Add enzyme: After shaking well, add 100 μL of enzyme conjugate to each well and gently shake to mix.

[0118] 7) Incubation: After sealing the plate with another sheet of sealing film, place it in a 37°C incubator for 45 minutes;

[0119] 8) Washing: Same as step 5.

[0120] 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 minutes;

[0121] 10) Measurement: Add 100 μL of stop solution to each well to terminate the reaction, gently shake to mix, and measure the absorbance A value of each well at a wavelength of 450 nm within 5 minutes.

[0122] 11) Result Interpretation: Using the concentration of the standard as X and the corresponding absorbance value A as Y, a quadratic polynomial fitting is performed.

[0123] Y = aX 2 +bX+c,

[0124] In the formula, Y represents the absorbance value A corresponding to the standard.

[0125] X represents the concentration of the standard, in ng / mL;

[0126] a, b, and c are all constants;

[0127] The concentration x of anti-L-type calcium channel antibody in the well of the coated plate is then calculated based on the fitted equation; the concentration of anti-L-type calcium channel antibody in the sample to be tested is calculated based on the following formula.

[0128] y = x * N;

[0129] In the formula, y represents the concentration of anti-L-type calcium channel antibody in the sample to be tested, (ng / mL);

[0130] x represents the concentration of anti-L-type calcium channel antibody in the well of the coated plate (ng / mL);

[0131] N is the dilution factor, N = 20.

[0132] Example 4

[0133] The above (enzyme-linked immunosorbent assay) kit detects samples positive for anti-L-type calcium channel antibodies.

[0134] Sixty-four serum samples that were positive for anti-L-type calcium channel antibodies were selected as needed for quantitative detection of anti-L-type calcium channel antibodies to determine the serum anti-L-type calcium channel antibody content.

[0135] The data in Table 4 show that the average coefficient of variation for the 64 samples was 13.02%, while the percentage of samples with a coefficient of variation below 15% was 67.19% (43 / 64). This indicates that the kit provides good repeatability for multiple tests.

[0136] Table 4. Repeatability of detection in positive samples against L-type calcium channel antibodies

[0137]

[0138]

[0139]

[0140] Example 5

[0141] The (enzyme-linked immunosorbent assay) kit is used to detect anti-L-type calcium channel antibodies and to aid in the diagnosis of viral myocarditis (VMC) and dilated cardiomyopathy (DCM).

[0142] 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.

[0143] The test results are shown in Table 5: The detection rate of anti-L-type calcium channel antibodies in patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM) using the kit was 89.74% (35 / 39); this indicates that the kit has good sensitivity and specificity for patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM), thus aiding in the diagnosis of these conditions and in the establishment of treatment plans. Table 5: Detection results of anti-L-type calcium channel antibodies in patients with viral myocarditis (VMC) and dilated cardiomyopathy (DCM)

[0144]

[0145]

[0146] 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 an L-type calcium channel 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: EVQLVESGPGLVKPSQSLSLTCSVTGYSITSAFYWNWIRQF PGNKLEWMGCISYDGSFHLNPSLKNRISVTRDTSKNLFFLRLNS VTSEDTAAYFCTRGTDYWGQGTTLTVSS; The amino acid sequence of the light chain is shown in SEQ ID No. 2: DIVMTQTPLSLFVSLGDQASISCRSSQSLVHSSGNTYLHWY LQKPGQSPKLLIYKLSSRFSGVPDRFSGSGSGTDFTLKISRVEAE DLGVYFCSQSTYVPWTFGGGTKLEIN.

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 the heavy chain CDR1 is SAFYWN. The amino acid sequence of the heavy chain CDR2 is CISYDGSFHLNPSLKN. The amino acid sequence of the heavy chain CDR3 is GTDY: 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 RSSQSLVHSSGNTYLH. The amino acid sequence of the light chain CDR2 is KLSSRFS. The amino acid sequence of the light chain CDR3 is SQSTYVPWT.

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 L-type calcium channel antigen, wherein the standards contain a standard solution of the humanized monoclonal antibody as described in claim 1.

6. The detection kit according to claim 4, characterized in that: The standard contained 1280 ng / mL of humanized monoclonal antibody against L-type calcium channels.

7. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, characterized in that: The L-type calcium channel antigen is shown in SEQ ID No. 3: VNENTRMYIPEENHQ.

8. The enzyme-linked immunosorbent assay (ELISA) kit according to any one of claims 5 to 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 L-type calcium channel antigen-coated plate is 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 L-type calcium channel 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 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) After measuring the concentration of the conjugate after dialysis in step 4), adjust the concentration to 10 μg / mL with carbonate buffer, add 100 μL / well to the microplate, and incubate at 2–8°C 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-L-type calcium channel autoantibodies using an enzyme-linked immunosorbent assay kit according to any one of claims 5 to 7, 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 is 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; Then, calculate the concentration x of anti-L-type calcium channel autoantibody in the sample well based on the fitted equation; calculate the concentration of anti-L-type calcium channel autoantibody in the sample to be tested based on the following formula; y = x * N; In the formula, y represents the concentration of anti-L-type calcium channel autoantibodies in the sample to be tested, in ng / mL; x represents the concentration of anti-L-type calcium channel autoantibodies in the wells of the coated plate, in ng / mL; N is the dilution factor.

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