A full-molecule IgG antibody, its preparation method and application

Abstract

A full-molecule IgG antibody, its preparation method, and its applications are disclosed. The amino acid sequences of the complementary antigen region (CDR) of the antibody's light chain are shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively; the amino acid sequences of the complementary antigen region (CDR) of the antibody's heavy chain are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively. The antibody prepared by this invention can effectively bind to EVA71, exhibiting good binding characteristics, and can be used to identify and block EVA71 infection.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals and relates to a human-mouse chimeric full-molecule IgG antibody against the capsid of enterovirus (EV) A71. It also relates to the DNA molecule, expression vector, host cell, and application of the above-mentioned full-molecule IgG antibody. Background Technology

[0002] Monoclonal antibody technology has played a significant role in disease diagnosis and treatment, greatly improving medical technology. However, conventional mouse or other animal-derived antibodies have been subject to ethical controversies due to their animal use, and their application in humans is severely limited because they can induce strong xenobiotic immune responses. Although there has been considerable development and application of anti-EVA71 antibodies based on mouse monoclonal antibodies in China, there has been no further application of genetically engineered or human-mouse chimeric antibodies. EVA71, enterovirus 71, is one of the main pathogens causing hand-foot-mouth disease (HFMD) in infants and young children. It still has a high infection rate in China, showing a regional epidemic trend. Therefore, clinical testing and available passive protective antibodies are also essential.

[0003] Human-mouse chimeric engineered antibody technology has matured and is an important method for altering the immunogenicity and functional characteristics of antibody molecules while maintaining their specific recognition characteristics. Currently published EVA71-related antibodies are primarily mouse-based and can be used for clinical testing and development. The antibody prepared in this invention can effectively bind to EVA71, exhibiting excellent binding properties, and there are currently no literature reports on human-mouse chimeric engineered antibodies with similar functions. Summary of the Invention

[0004] Technical problem to be solved: The present invention aims to provide a human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, its preparation method and application, which can recognize and block EVA71 infection.

[0005] Technical solution: A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG, wherein the amino acid sequences of the antigen complementary region (CDR) of the antibody light chain are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; and the amino acid sequences of the antigen complementary region (CDR) of the antibody heavy chain are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0006] A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG, wherein the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.3 and the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.4.

[0007] A human-mouse chimeric anti-EVA71 capsid protein full VP1 molecule IgG, the nucleic acid sequence of the antibody light chain variable region is shown in SEQ ID NO.1, and the nucleic acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO.2.

[0008] A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, the light chain amino acid sequence of which is shown in SEQ ID NO.11 and the heavy chain amino acid sequence of which is shown in SEQ ID NO.12.

[0009] Plasmids containing the above nucleic acid sequences.

[0010] Eukaryotic cells containing the plasmid.

[0011] The above-mentioned human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG was used in the preparation of a kit for detecting EV71.

[0012] The above-mentioned human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG is used in the preparation of diagnostic or therapeutic drugs to block EV71 infection.

[0013] A test kit containing the above-mentioned human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG.

[0014] A drug for blocking EV71 infection contains the above-mentioned human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG.

[0015] Beneficial effects: The antibody prepared by this invention can effectively bind to EVA71, showing good binding characteristics, and can be used to identify and block EVA71 infection. Attached Figure Description

[0016] Figure 1 This is an SDS-PAGE assay image. M: Protein Marker; 1: Purified and concentrated antibody.

[0017] Figure 2 This is a schematic diagram of an enzyme-linked immunosorbent assay (ELISA).

[0018] OD value 2.845 1.978 1.162 0.507 0.322 0.172 0.114

[0019] The purified antibody was diluted 20,000 times and then serially diluted 2 times, with a cutoff value of 0.015. The final detection titer was greater than 1:1,280,000.

[0020] Figure 3 This is a schematic diagram of a standard antigen test.

[0021] OD 1.784 1.101 0.481 0.254 0.114

[0022] Figure 4 This is a schematic diagram of Western Blot detection; M: Protein Marker; 1: VP1-expressing 293T cells; 2: VP1-expressing cell supernatant; 3: Control 293T cells; 4: Control cell supernatant.

[0023] Figure 5 This is a schematic diagram of immunofluorescence detection (293T transfection). The purified humanized antibody was used to transfect VP1 cells into 293T cells, and immunofluorescence detection was performed at a working concentration of 1 g / mL. Images were taken using a fluorescence microscope. The images show the TRITC fluorescence channel, the DAPI nuclear staining channel, and the fusion image. Detailed Implementation

[0024] 1. Culture and preparation of murine anti-EV A71 capsid protein VP1 hybridoma cells;

[0025] 2. Amplification, sequencing, analysis, and identification of the murine anti-EV A71 antibody sequence;

[0026] 3. Preparation, expression, and purification of human-mouse chimeric anti-EV A71 full-molecule IgG;

[0027] 4. Characterization of human-mouse chimeric anti-EV A71 full-molecule antibody;

[0028] 5. Anti-EV A71 full-molecule IgG is used for detection.

[0029] Example 1. Culture and preparation of mouse anti-EV A71 capsid protein VP1 hybridoma cells

[0030] Based on the gene sequence of EVA71, the corresponding sequence of VP1 was synthesized and cloned into a prokaryotic expression vector, and the corresponding antigen was purified by prokaryotic expression.

[0031] The expressed recombinant protein was used as an immunogen to immunize pure BALB / c mice via subcutaneous injection in the abdomen, 200 μg each time, for a total of five times. A booster immunization was administered intraperitoneally seven days before the final immunization, which was prior to cell fusion. On the day of fusion, mouse spleens were harvested and a single-cell suspension was prepared in DMEM medium (GIBCO, USA). Spleen cells and SP2 / 0 mouse myeloma cells were fused in the presence of 50% PEG (pH 8.0). The cells were cultured in HAT selective medium (98 mL DMEM, 1 mL HT stock solution, 1 mL A stock solution) for 7 days, then the medium was changed to HT medium (99 mL DMEM, 1 mL HT).

[0032] Example 2. Screening, preparation and identification of murine anti-HEV antibodies

[0033] Hybridoma cells were screened using enzyme-linked immunosorbent assay (ELISA) based on their growth status, as detailed below. Cells from positive wells were then cultured again for subcloning. After three subcloning cycles, once the supernatant from all wells showed positive results for anti-EVA71, several wells were expanded for further culture and some were cryopreserved.

[0034] The ELISA method for screening anti-EVA71 positive individuals is as follows:

[0035] (1) Purify the prokaryotic antigen, coat the 96-well ELISA plate, dilute with coating buffer (0.1M carbonate buffer, pH 9.6) to 2 μg / mL, add 100 μL to each well, and incubate overnight at 4°C;

[0036] (2) Wash 5 times with PBST washing buffer (PBS containing 0.05% Tween), then add 5% BSA (200 μL / well) to block, incubate at room temperature for 2 h, discard the blocking solution and store at 4℃;

[0037] (3) Add 100 μL of hybridoma cell culture supernatant to each well, use the serum of immunized mice as a positive control (1:1000 dilution) and the serum of blank mice as a negative control (1:1000 dilution), incubate at 37°C for 1 h, discard the detection liquid, and wash 5 times with PBST washing solution.

[0038] (4) Add 100 μL / well of goat anti-mouse Ig-HRP secondary antibody (Thermo) diluted at 1:5000 to the well, incubate at 37°C for 1 h, discard the detection liquid, and wash 5 times with PBST;

[0039] (5) Add 100 μL of peroxidase substrate chromogenic solution per well, and stop the reaction with 2M sulfuric acid after 15 minutes at room temperature. Detect using a full-wavelength microplate reader (Thermo Labsystems, USA), using dual wavelengths of 450 nm / 630 nm. A positive clone is defined as a result with an OD value greater than 1.0 of normal mouse serum.

[0040] The purified antibody was subjected to antibody subtype identification using the ISO-2KT monoclonal antibody subtype identification kit from Sigma-Aldrich, USA. The results showed that the subtype of the anti-HEV antibody was IgG2a.

[0041] Example 2. Amplification, sequencing, analysis and identification of mouse anti-EV A71 antibody sequence

[0042] Hybridoma cells were revived, and total RNA was extracted according to the Trizol Reagent Kit instructions. cDNA was obtained by RT-PCR. Based on statistical data from the BLAST database, 19 VH forward and 17 Vκ forward primers, 4 VH reverse and 3 Vκ reverse primers were designed. The primer sequences are as follows:

[0043] Vκforward primers

[0044] Vκ-1: 5'-GGGCCCAGGCGGCCGAGCTCGAYATCCAGCTGACTCAGCC-3'

[0045] Vκ-2: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCWCCCAGTC-3'

[0046] Vκ-3: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTGMTMACTCAGTC-3'

[0047] Vκ-4: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTGYTRACACAGTC-3'

[0048] Vκ-5: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTRATGACMCAGTC-3'

[0049] Vκ-6: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTMAGATRAMCCAGTC-3'

[0050] Vκ-7: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTCAGATGAYDCAGTC-3'

[0051] Vκ-8: 5'-GGGCCCAGGCGGCCGAGCTCGAYATYCAGATGACACAGAC-3'

[0052] Vκ-9: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGTTCTCAWCCAGTC-3'

[0053] Vκ-10: 5'-GGGCCCAGGCGGCCGAGCTCGAYATTGWGCTSACCCAATC-3'

[0054] Vκ-11:5’-GGGCCCAGGCGGCCGAGCTCGAYATTSTRATGACCCARTC-3’

[0055] Vκ-12:5’-GGGCCCAGGCGGCCGAGCTCGAYATTKTGATGACCCARAC-3’

[0056] Vκ-13:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACBCAGKC-3’

[0057] Vκ-14:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATAACYCAGGA-3’

[0058] Vκ-15:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACCCAGWT-3’

[0059] Vκ-16:5’-GGGCCCAGGCGGCCGAGCTCGAYATTGTGATGACACAACC-3’

[0060] Vκ-17:5’-GGGCCCAGGCGGCCGAGCTCGAYATTTTGCTGACTCAGTC-3’

[0061] Vκ3’reverse primers

[0062] VκR1:5’-AGATGGTGCAGCCACAGTTCGTTTKATTTCCAGYTTGGTCCC-3’

[0063] VκR2:5’-AGATGGTGCAGCCACAGTTCGTTTTATTTCCAACTTTGTCCC-3’

[0064] VκR3:5’-AGATGGTGCAGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC-3’

[0065] VH 5’forward primers

[0066] VH 1:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTRMAGCTTCAGGAGTC-3’

[0067] VH 2:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTBCAGCTCAGCAGTC-3’

[0068] VH 3:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGCTGAAGSASTC-3’

[0069] VH 4:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCARCTGCAACARTC-3’

[0070] VH 5:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCAGCTBCAGCARTC-3’

[0071] VH 6:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTYCARCTGCAGCAGTC-3’

[0072] VH 7:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCACGTGAAGCAGTC-3’

[0073] VH 8:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAASSTGGTGGAATC-3’

[0074] VH 9:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAWGYTGGTGGAGTC-3’

[0075] VH 10:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAGSKGGTGGAGTC-3’

[0076] VH 11:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCAMCTGGTGGAGTC-3’

[0077] VH 12:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGCTGATGGARTC-3’

[0078] VH 13:5’-GCTGCCCAACCAGCCATGGCCCTCGAGGTGCARCTTGTTGAGTC-3’

[0079] VH 14: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTRAAGCTTCTCGAGTC-3'

[0080] VH 15: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAARSTTGAGGAGTC-3'

[0081] VH 16: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTTACTCTRAAAGWGTTSG-3'

[0082] VH 17: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTCCAACTVCAGCARCC-3'

[0083] VH 18: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAACTTGGAAGTGTC-3'

[0084] VH 19: 5'-GCTGCCCAACCAGCCATGGCCCTCGAGGTGAAGGTCATCGAGTC-3'

[0085] VH 3'reverse primers

[0086] VH R1: 5'-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGTGGT-3'

[0087] VH R2: 5'-CGATGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGT-3'

[0088] VH R3: 5'-CGATGGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAGT-3'

[0089] VH R4: 5'-CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACTGAGGT-3'

[0090] Using the primers described above, the VH and VL genes were amplified under the following conditions: 95℃ for 4 min, 95℃ for 30 s, 56℃ for 30 s, and 72℃ for 30 s, for a final extension at 72℃ for 10 min. The amplified gene fragments were recovered and purified by electrophoresis, ligated into pMD-18T, transformed into E. coli Top10F', and sequenced to obtain the light and heavy variable region sequences.

[0091] (1) PCR amplification of antibody genes;

[0092] The reaction system is as follows:

[0093]

[0094] The reaction conditions are as follows:

[0095]

[0096] (2) 2% agarose gel electrophoresis, observe the target band under ultraviolet light, and cut and recover the gel;

[0097] (3) Purify the target DNA fragment using a gel extraction kit and elute with deionized water;

[0098] (4) Double enzyme digestion of IgG expression plasmid;

[0099] The IgG expression plasmids pFUSE-CHIg-hG1 and pFUSE-CLIg-hk (purchased from Invivogen) contain the base coding sequences of the heavy and light chain (Kappa) constant regions of human IgG1.

[0100] Double digestion of the pFUSE-CHIg-hG1 and pFUSE-CLIg-hk template vectors;

[0101] The reaction system is as follows:

[0102]

[0103] The reaction conditions were: enzyme digestion overnight at 37°C.

[0104] b. 1% agarose gel electrophoresis, followed by UV-cut gel recovery;

[0105] c. Purify the target DNA fragment using a gel extraction kit, followed by elution with deionized water.

[0106] (5) Infusion PCR recombinant expression plasmid;

[0107] VHR 5'-GCCCTTGGTGGATGCTGAAGAGACAGTGAC-3' VKF 5'ACAGACGCTCGCTGCGACATTGTGATGACCCAG-3' VKR 5'-TGCAGCCACCGTACGTTTTATTTCCAGCTT-3'

[0108] The corresponding sequence was amplified, and the reaction system and target band recovery are described in Example 2. The target gene was then recombinantly cloned.

[0109] The reaction system is as follows:

[0110]

[0111] The reaction conditions were: incubation at 50°C for 15 minutes.

[0112] Transform competent bacteria with 5 μL of reaction solution, plate them on plates with the appropriate antibiotics, and send clones for sequencing the next day. Preserve the bacterial strain of the correctly sequenced clones and expand the culture, extract plasmids, and determine their concentration and quality.

[0113] Example 3. Preparation, expression, and purification of human-mouse chimeric anti-EV A71 full-molecule IgG

[0114] (1) Transfection and expression of human-mouse chimeric anti-EV A71 full-molecule IgG

[0115] a. Take 50 μg of the recombinant heavy chain plasmid into 1 mL of Opti-MEM medium, take 50 μg of the light chain plasmid into 1 mL of Opti-MEM medium, and take 200 μL of 293Fectin into 2.8 mL of Opti-MEM medium. Let the above three mixtures stand at room temperature for 5 min.

[0116] b. After thoroughly mixing the two plasmid mixtures, add 500 μL of Opti-MEM medium and mix well. Then, directly add the 293Fectin transfection reagent mixture and mix well. Let it stand for 20 min. During this time, treat the 293F cells by centrifuging them and resuspending them in 293F Expression Medium. Then, count the cells and calculate the cell viability ratio using trypan blue. Take 1.00 × 10⁻⁶ cells... 8 One cell was placed in a culture flask and brought to a final volume of 94 mL with 293F Expression Medium.

[0117] c. After 20 minutes, add 6 mL of the DNA and 293Fectin complex to the prepared 293F cells;

[0118] d. Place the cells in a shaker incubator and culture them under the following conditions: 8% CO2, 120 rpm, 37°C. Collect the cell supernatant after 6 days.

[0119] (2) Purification of human-mouse chimeric anti-EV A71 full-molecule IgG

[0120] The collected cell culture supernatant was filtered through a 0.45 μm filter membrane, simultaneously filtering out the equilibration buffer and elution buffer. Purification was performed using the AKATA P100 protein purification system following standard Protein A purification procedures, with loading at a flow rate of 1 mL / min and elution at a flow rate of 1.5 mL / min. The purified protein was then identified by SDS-PAGE; the results are shown below. Figure 1 The expression level was measured to be 2.7 mg / 100 mL.

[0121] Example 4. Enzyme-linked immunosorbent assay (ELISA) of human-mouse chimeric anti-EV A71 full-molecule antibody to detect antibody specificity and titer.

[0122] Dilute EV A71 with coating buffer (0.1M carbonate buffer, pH 9.6) VP1 recombinant protein was added to 2 μg / mL to coat EIA high affinity 96-well plates, 100 μL per well, and incubated overnight at 4°C. The liquid was discarded, and the plates were washed 5 times with PBST, then blocked with PBST (PBS containing 0.5% Tween 20) and 5% BSA-wash buffer, and incubated at room temperature for 2 h. The liquid was discarded and the plates were stored at 4°C. For detection, after equilibration at room temperature for half an hour, 100 μL of serially diluted anti-HEV full-molecule IgG (1 μg / mL starting concentration, 7 concentration serial dilutions) was added to each well, and incubated at 37°C for 1 h. The liquid was discarded, and the plates were washed 5 times with PBST, then 100 μL of goat anti-human secondary antibody (1:2000 dilution) was added to each well, and incubated at 37°C for 1 h. The liquid was discarded, and the plates were washed 5 times with PBST, then peroxidase substrate chromogenic solution was added, and the reaction was stopped with 2M sulfuric acid after 15 min at 37°C. The absorbance of the protein was then measured.

[0123] The results are as follows Figure 2 The results showed that the humanized antibody could exhibit a significant antigen-antibody reaction with the recombinant EVA71 VP1 protein.

[0124] Example 5. Application of full-molecule anti-EV A71 IgG in detection

[0125] (1) ELISA detection application of EVA71 capsid protein VP1

[0126] 1) Immunize rabbits with VP1 protein three times, monitor the titer, collect rabbit serum, purify and prepare rabbit immunoglobulin, and adjust the concentration to 1 mg / mL;

[0127] 2) EIA high-affinity plates were coated with 2 μg / mL rabbit polyclonal antibody and incubated overnight at 4°C;

[0128] 3) After routine washing, add 3% BSA and seal at 4°C overnight;

[0129] 4) Discard the supernatant and store at 4℃ for later use;

[0130] 5) Add 100 μl of the prepared sample (prokaryotic antigen preparation, gene expression product and clinical sample), and incubate at 37°C for 1 hour;

[0131] 6) After routine plate washing, add 0.1 μg / mL of genetically engineered antibody and incubate at 37°C for 1 hour;

[0132] 7) After routine plate washing, add sheep human HRP-labeled antibody and incubate at 37°C for 1 hour;

[0133] 8) Perform a standard plate wash, add the developing solution, and develop for 15 minutes;

[0134] 9) After adding the stop solution, perform absorbance detection at 450 / 520nm wavelength.

[0135] The detection results showed that the sensitivity of this application for detecting prokaryotic expressed proteins reached 15±3.4 ng / mL. (See attached image.) Figure 3 The detection sensitivity of EV71 transfection supernatant reached over 1:6400. Pharyngeal swab samples were collected from patients with mouth and foot disease, and the positive rate of clinical samples reached 75%. (2) Application of Western blot detection of EV A71 capsid protein VP1

[0136] 1) Collect transfected 293T cells and supernatant;

[0137] 2) Add protein lysis buffer and loading buffer separately to process the samples;

[0138] 3) After loading the PAGE sample, the proteins were separated by electrophoresis and then transferred to a PVDF membrane;

[0139] 4) Standard 5% skim milk powder is sealed at room temperature for 2 hours;

[0140] 5) Add 1 μg / mL of genetically engineered antibody and incubate at 37°C for 1 hour;

[0141] 6) Wash the membrane as usual, add sheep human HRP-labeled antibody (1:5000), and incubate at 37°C for 1 hour;

[0142] 7) Perform routine washing of the membrane, add the chemiluminescent substrate, and develop the color for 1 minute;

[0143] 8) Perform chemiluminescence imaging.

[0144] The results showed that the antibody effectively recognized the expressed EV71 antigen, with the molecular weight as expected and good specificity. The corresponding antigen was effectively detected in both the expression supernatant and cells. (See Figure 4.)

[0145] (3) Application of Western blot detection of EV A71 capsid protein VP1

[0146] 293T cell lines transfected with the EV A71 VP1 plasmid were cultured on glass slides. When cell confluence reached over 90%, the cell slides were removed and fixed in ice-cold acetone. The acetone solution was washed off with PBST, and 100 μL of humanized antibody (1 μg / mL) was added. The slides were incubated at 37°C for 1 h, washed five times with PBST, and then 100 μL of TRTIC-labeled goat-human antibody (1:5000 dilution) was added. The slides were incubated at 37°C for 1 h, washed five times with PBST, and then mounted with DAPI. The slides were then observed under a confocal microscope at a specific wavelength (see [link to article]). Figure 5 ).

Claims

1. A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, characterized in that, The amino acid sequences of the antibody light chain antigen complementary region CDR are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively; the amino acid sequences of the antibody heavy chain antigen complementary region CDR are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

2. A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, characterized in that, The amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.3, and the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.

4.

3. A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, characterized in that, The nucleic acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.1, and the nucleic acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.

2.

4. A human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody, characterized in that, The light chain amino acid sequence of the antibody is shown in SEQ ID NO.11, and the heavy chain amino acid sequence is shown in SEQ ID NO.

12.

5. The use of the human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody as described in claim 1 in the preparation of a kit for detecting EVA71.

6. A test kit, characterized in that, Contains the human-mouse chimeric anti-EVA71 capsid protein full-molecule IgG antibody as described in claim 1.

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