Antibodies against coxsackievirus a16 and methods of making and using the same

Abstract

The present application relates to the technical field of antibodies, in particular to an antibody against coxsackievirus A16 and a preparation method and application thereof.The heavy chain complementarity determining region CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof provided in the present application have the amino acid sequences shown as SEQ ID NO.1-3, and the light chain complementarity determining region CDR1, CDR2 and CDR3 have the amino acid sequences shown as SEQ ID NO.4-6.The antibody or antigen binding fragment thereof can specifically bind to and neutralize CV-A16 strain and its antigen, has good specificity, has a good protective effect on animals infected with CV-A16, and can be used for evaluating CV-A16 vaccine antigenicity and immunogenicity, diagnosing CV-A16 infection and developing CV-A16 prevention or treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibodies, in particular to an antibody against Coxsackievirus A16 and a preparation method and application thereof. BACKGROUND

[0002] Hand, foot and mouth disease (HFMD) is a serious infectious disease affecting the health of infants and young children. Coxsackievirus A16 (CV-A16) and Enterovirus A71 (EV-A71) are the main pathogens causing hand, foot and mouth disease. Vaccination is the main means to control the epidemic of infectious diseases. Most of the CV-A16 vaccines currently in the research and development stage are inactivated vaccines.

[0003] Antibodies can be used not only for clinical diagnosis and treatment, but also for vaccine and antigen detection and evaluation. Among them, monoclonal antibodies have the characteristics of high targeting, low toxicity, etc., and are widely used in clinical diagnosis, treatment and vaccine evaluation. Quantitative detection of antigen content is one of the important methods for quality control of vaccine production process and final product, and the establishment of accurate and rapid antigen detection method can effectively promote the research and development process of CV-A16 vaccine. The CA16 monoclonal antibody is an important part to ensure the specificity and accuracy of detection. Therefore, the development of CA16 antibody with excellent performance is of great significance for the development and detection of CV-A16 vaccine and the clinical diagnosis and prevention of CV-A16 infection. SUMMARY

[0004] The purpose of the present application is to provide an antibody against Coxsackievirus A16 and a preparation method and application thereof.

[0005] Specifically, the present application provides the following technical solutions:

[0006] In the first aspect, the present application provides an antibody or an antigen binding fragment thereof, wherein the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or the antigen binding fragment thereof are as shown in SEQ ID NO. 1-3, and / or the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or the antigen binding fragment thereof are as shown in SEQ ID NO. 4-6.

[0007] In the present application, the antigen binding fragment refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, or can compete with the full-length antibody for specific binding to the antigen.

[0008] In some embodiments of the present application, the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen binding fragment thereof are as shown in SEQ ID NOs. 1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR3 are as shown in SEQ ID NOs. 4-6.

[0009] The above antibody or antigen binding fragment thereof provided by the present application can specifically neutralize the CV-A16 strain, and has good specificity.

[0010] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 7 or has at least 80% homology with the amino acid sequence as shown in SEQ ID NO. 7; and / or, the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 8 or has at least 80% homology with the amino acid sequence as shown in SEQ ID NO. 8.

[0011] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 8.

[0012] In some embodiments of the present application, in the case of having the above heavy chain and light chain complementarity determining regions CDR1, CDR2, CDR3, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the amino acid sequence as shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the amino acid sequence as shown in SEQ ID NO. 8.

[0013] In some embodiments of the present application, the antibody or antigen binding fragment thereof further comprises a signal peptide sequence at the N-terminus of the heavy chain variable region.

[0014] In some embodiments of the present application, the signal peptide at the N-terminus of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 9.

[0015] In some embodiments of the present invention, the amino acid sequence of the antibody or antigen-binding fragment thereof further comprises a signal peptide sequence at the N-terminus of the light chain variable region.

[0016] In some embodiments of the present invention, the signal peptide at the N-terminus of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.10.

[0017] In the present invention, the antibody can be a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0018] The monoclonal antibody may be an animal-derived antibody (eg, a mouse-derived antibody), a chimeric antibody, or a humanized antibody.

[0019] In the present invention, the antigen-binding fragment may be Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment or single-chain antibody.

[0020] In some embodiments of the invention, the antibody further comprises a heavy chain constant region.

[0021] In some embodiments of the invention, the heavy chain of the antibody is IgG3.

[0022] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.11.

[0023] In some embodiments of the invention, the antibody further comprises a light chain constant region.

[0024] In some embodiments of the present invention, the light chain of the antibody is Kappa.

[0025] In some embodiments of the present invention, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.12.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0027] Based on the amino acid sequence of the above-mentioned antibody or antigen-binding fragment thereof, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof is not unique. All nucleic acid molecules capable of encoding and producing the above-mentioned antibody or antigen-binding fragment thereof are within the scope of protection of the present invention.

[0028] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the above-mentioned antibody is shown as SEQ ID NO. 13, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the above-mentioned antibody is shown as SEQ ID NO. 14.

[0029] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the above-mentioned antibody is shown as SEQ ID NO. 15, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the above-mentioned antibody is shown as SEQ ID NO. 16.

[0030] In some embodiments of the present application, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the above-mentioned antibody containing a signal peptide is shown as SEQ ID NO. 17, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the above-mentioned antibody containing a signal peptide is shown as SEQ ID NO. 18.

[0031] In a third aspect, the present application provides a biological material comprising the above-mentioned nucleic acid molecule; the biological material is an expression cassette, a vector or a host cell.

[0032] The above-mentioned expression cassette can be a recombinant DNA obtained by operably linking the above-mentioned nucleic acid molecule with a promoter.

[0033] The above-mentioned vector includes but is not limited to a plasmid vector, a bacteriophage vector, a viral vector, an artificial chromosome vector, etc.

[0034] The above-mentioned host cell includes a microbial cell, an animal cell or a cell line. The microbial cell includes but is not limited to Escherichia coli, yeast, etc., the animal cell includes but is not limited to an insect cell, and the cell line includes but is not limited to a CHO cell, a 293T cell, etc.

[0035] In a fourth aspect, the present application provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or antigen binding fragment thereof with a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

[0036] In a fifth aspect, the present application provides a preparation method of the above-mentioned antibody or antigen binding fragment thereof, which comprises culturing a host cell capable of expressing the above-mentioned antibody or antigen binding fragment thereof, and isolating the above-mentioned antibody or antigen binding fragment thereof.

[0037] In a sixth aspect, the present application provides any one of the following applications of the above-mentioned antibody or antigen binding fragment thereof, the above-mentioned nucleic acid molecule, the above-mentioned biological material or the above-mentioned antibody conjugate:

[0038] (1) an application in the preparation of a product for detecting the presence or level of Coxsackievirus A16 in a sample;

[0039] (2) use in the manufacture of a product for diagnosing Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection;

[0040] (3) use for detecting the presence or level of Coxsackievirus A16 in a sample for non-diagnostic and therapeutic purposes;

[0041] (4) use for detecting the antigenicity or immunogenicity of a Coxsackievirus A16 vaccine;

[0042] (5) use for quality control of Coxsackievirus A16 vaccine production;

[0043] (6) use for detecting the specificity or content of Coxsackievirus A16 antigen;

[0044] (7) use in the manufacture of a product for neutralizing the virulence of Coxsackievirus A16 in a sample;

[0045] (8) use in the manufacture of a medicament for preventing or treating Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection.

[0046] In the uses described in (1) and (3) above, detecting the presence of Coxsackievirus A16 in a sample refers to detecting whether a sample contains Coxsackievirus A16, and detecting the level of Coxsackievirus A16 refers to detecting the content of Coxsackievirus A16 in a sample.

[0047] The sample can be a sample from a living human or animal (including blood, excrement, oral and nasal secretions, etc.), or a sample of cells cultured in vitro.

[0048] In the uses described in (1) and (2) above, the product can be a detection reagent or a kit.

[0049] In the use described in (3) above, the sample can be a sample of cells cultured in vitro, a vaccine, or the like, and is not a sample from a living human or animal.

[0050] In the uses described in (2) and (8) above, the disease caused by Coxsackievirus A16 infection includes hand, foot and mouth disease, etc.

[0051] In the use described in (4) above, the detection of immunogenicity specifically refers to detecting the performance of an animal's immune response induced by a Coxsackievirus A16 vaccine, including evaluation of the animal's humoral immune function (e.g., neutralizing antibodies and their levels, antibody affinity), etc.

[0052] In the application of the above (5), the quality control of the Coxsackievirus A16 vaccine specifically refers to detecting whether the antigen quality, content, stability and the like of the Coxsackievirus A16 vaccine are qualified.

[0053] In the application, the detection method can use enzyme-linked immunosorbent (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography and the like. The antibody or the antigen binding fragment thereof provided in the application can be used as a binding antibody in the above detection methods, and is used for detection of the Coxsackievirus A16 antigen.

[0054] In a seventh aspect, the application provides a kit comprising the antibody or the antigen binding fragment thereof or the antibody conjugate.

[0055] The above kit has any one of the following uses:

[0056] (1) detecting the presence or level of the Coxsackievirus A16 in a sample;

[0057] (2) diagnosing Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection;

[0058] (3) detecting the antigenicity and immunogenicity of the Coxsackievirus A16 vaccine;

[0059] (4) quality control of production of the Coxsackievirus A16 vaccine;

[0060] (5) detecting the specificity and content of the Coxsackievirus A16 antigen.

[0061] In addition to comprising the antibody or the antigen binding fragment thereof or the labeled antibody, the above kit can further comprise a second antibody carrying a detectable label to detect the antibody or the antigen binding fragment thereof of the application. The detectable label includes but is not limited to an enzyme, a radioisotope, a fluorescent dye and the like.

[0062] In an eighth aspect, the application provides a drug comprising the antibody or the antigen binding fragment thereof.

[0063] The above drug has any one of the following uses:

[0064] (1) preventing Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection;

[0065] (2) treating Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection.

[0066] In addition to comprising the antibody or the antigen binding fragment thereof, the above drug can further comprise a pharmaceutically acceptable carrier, an excipient or other active ingredients.

[0067] In a ninth aspect, the present application provides a method for detecting the presence or level of Coxsackievirus A16 in a sample, which comprises detecting the presence or level of Coxsackievirus A16 in a sample using the antibody or antigen-binding fragment thereof of the present application. The method can be used for diagnostic purposes (the sample is from a living human or animal) or non-diagnostic purposes (the sample is a cell sample cultured in vitro rather than a sample from a living human or animal).

[0068] In a tenth aspect, the present application provides a method for diagnosing Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection, which comprises diagnosing Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection using the antibody or antigen-binding fragment thereof of the present application.

[0069] In an eleventh aspect, the present application provides a method for neutralizing the virulence of Coxsackievirus A16 in a sample, which comprises contacting a sample comprising Coxsackievirus A16 with the antibody or antigen-binding fragment thereof of the present application. The method can be used for therapeutic purposes or non-therapeutic purposes (the sample is a cell sample cultured in vitro rather than a sample from a living human or animal).

[0070] In a twelfth aspect, the present application provides a method for preventing or treating Coxsackievirus A16 infection or a disease caused by Coxsackievirus A16 infection (including hand, foot and mouth disease, etc.), which comprises administering to a subject a prophylactically or therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present application or a medicament comprising the antibody or antigen-binding fragment thereof.

[0071] In a thirteenth aspect, the present application provides a quality control method for Coxsackievirus A16 vaccine production, which comprises the step of detecting the Coxsackievirus A16 antigen contained in the vaccine using the antibody or antigen-binding fragment thereof of the present application.

[0072] The beneficial effects of the present application include at least the following aspects:

[0073] The antibody or antigen-binding fragment thereof provided by the present application can specifically bind to the CV-A16 strain and its antigen, has good specificity, has no cross-binding reaction with other types of enterovirus (EV-A71 / CV-A6 / CV-A10) antigens and VERO cell host proteins, and can be used for the evaluation of CV-A16 vaccine antigenicity and immunogenicity, the specific detection and quantitative analysis of CV-A16 virus antigens, and the diagnosis of CV-A16 infection, and further developed into an antigen detection kit, which is of great significance for the development, evaluation of vaccines and the clinical diagnosis of CV-A16 infection.

[0074] The antibody or antigen binding fragment thereof provided by the application can specifically neutralize the CV-A16 strain, and can neutralize the CV-A16 virus (100 CCID 50 / 0.05ml) so as to be unable to infect cells, and has no cross-neutralization reaction with other types of enterovirus (EV-A71 / CV-A6 / CV-A10); the antibody can competitively inhibit 96% of the neutralizing antibody components in human natural infection serum, has a strong immune competition advantage, and provides a basis for development of therapeutic or prophylactic antibody drugs; the antibody has a good protective effect on animals infected with CV-A16, and the protection effect shows a dose-effect relationship. In a BALB / c mouse model, 16 μg of the antibody has a therapeutic effect on BALB / c mice infected with the CV-A16 virus, and can be applied to development of a CV-A16 prevention or treatment drug, and has important significance for treatment of CV-A16 infection and diseases related thereto. BRIEF DESCRIPTION OF DRAWINGS

[0075] In order to more clearly illustrate the technical solutions in the application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0076] Figure 1 It is a comparison chart of the solid virus particles and the hollow virus particles identified by electron microscopy after the sucrose density gradient centrifugation in Example 1 of the application, wherein the upper chart is the hollow particle, and the lower chart is the solid particle.

[0077] Figure 2 It is the SDS-PAGE detection result in Example 3 of the application.

[0078] Figure 3 It is the neutralizing antibody titer determination result in Example 3 of the application.

[0079] Figure 4 It is the fluorescence antibody staining (objective lens 20x) in Example 3 of the application, wherein the left chart is the CV-A16 virus infection, and the right chart is the cell control.

[0080] Figure 5 It is the competitive inhibition test result of the monoclonal antibody and the human serum in Example 3 of the application.

[0081] Figure 6 It is the enzyme-labeled antibody reaction curve in Example 5 of the application.

[0082] Figure 7 It is the linear correlation detection result in Example 7 of the application.

[0083] Figure 8 Specificity test results in Example 7 of the present application.

[0084] Figure 9 Survival curve in Example 8 of the present application. DETAILED DESCRIPTION

[0085] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without any creative effort fall within the protection scope of the present application.

[0086] Preparation of CV-A16 antigen in Example 1

[0087] Virus culture: take one CV-A16 strain, dilute it by 1:10000, inoculate it into four 10-layer cell factories covered with single-layer Vero cells, 2L per factory, and place it in a 35°C constant temperature incubator for 3-5 days. When the lesion reaches 100%, the virus liquid is harvested. The virus harvest liquid is divided into centrifuge cups, centrifuged at 6000 rpm for 40 min, and the centrifugal supernatant is harvested to obtain the CV-A16 centrifugal supernatant.

[0088] Ultrafiltration concentration: the centrifugal supernatant is ultrafiltrated and concentrated with a 300KD membrane. The membrane is washed twice with 10mmol / L PBST solution, and the washing liquid and the unfiltered liquid are combined as the ultrafiltration product.

[0089] Sucrose density gradient centrifugation: the ultrafiltration product is diluted 3.5 times with 10mmol / L PBS solution (0.15mol / L NaCl) and subjected to gradient centrifugation. The sucrose concentration is 40%-55%. Centrifuge at 30000 rpm for 15-18h, 35ml per tube, and collect 24 tubes. Take samples for SDS-PAGE detection, combine the empty and solid particles according to the detection results, and identify the empty and solid particles with an electron microscope.

[0090] Comparison chart of solid virus particles and empty virus particles identified by electron microscope after sucrose density gradient centrifugation Figure 1 .

[0091] Preparation of monoclonal antibody in Example 2

[0092] Hybridoma cell line preparation: The hollow particles and solid particles were collected and desugared, and then mixed at a volume ratio of 1:1 to obtain the CV-A16 virus purification liquid. NIH mice were immunized, 5 mice for each type. The mice were immunized by subcutaneous injection at multiple sites on the back at 0, 2, 4, 6, and 8 weeks. The immunization dose was 30 μg / mouse for the first injection with Freund's complete adjuvant, 15 μg / mouse for the second and third injections with Freund's incomplete adjuvant, and 15 μg / mouse for the fourth injection without adjuvant, injected into the tail vein. Blood sampling and testing: blood was collected from the mice one week after the second and third injections for indirect enzyme-linked immunoassay to determine the antibody titer. When the antibody titer reached 10 4 The mice were given a fourth intraperitoneal injection for booster immunization.

[0093] Cell fusion: cell fusion was performed using an electric fusion method. The average fusion efficiency was that about 2500 spleen cells could fuse to produce one hybridoma cell.

[0094] Screening and verification: the supernatant of the fused cells was preliminarily screened using an indirect ELISA method, and the supernatant positive to the immunogen was selected. All the positive clones obtained in the preliminary screening stage were confirmed by indirect ELISA. The positive clones confirmed in the screening were verified for neutralizing activity using a neutralizing antibody determination method. Positive clones with neutralizing activity and high binding titer were screened.

[0095] Subcloning, expansion culture, and cryopreservation: the positive parent clone cells were transferred to a 24-well plate for expansion culture, and the cell supernatant was collected for re-verification. Finally, the positive parent clones were subcloned twice using a limiting dilution method, and screened using an indirect ELISA method and a neutralizing antibody determination method. The positive clone strains confirmed after the second subcloning were expanded, and mice were immunized to prepare ascites.

[0096] Example 3 Purification and identification of monoclonal antibody

[0097] A monoclonal antibody was obtained through screening, which was named monoclonal antibody 24G9. The purification and performance identification results of the monoclonal antibody 24G9 are as follows:

[0098] Antibody purification: the ascites prepared from the immunized mice in Example 2 was centrifuged at 2-8°C and 4000-8000 r / min for 5-15 minutes, and the supernatant was filtered through a 0.45 μm filter membrane. The antibody was purified by Protein A / G affinity chromatography, and the purified antibody was stored in phosphate buffered saline (PBS) by dialysis. The protein content was 0.881 mg / ml detected by the Lowry method. The purity of the purified monoclonal antibody was detected, and the titer was determined.

[0099] SDS-PAGE purity determination: analysis showed that the heavy chain and light chain of the monoclonal antibody 24G9 were about 50 KD and 25 KD, respectively, as shown in Figure 1. Figure 2indicated.

[0100] Binding antibody titer assay: EV-A71, CV-A16, CV-A6, CV-A10 virus purified solution and Vero cell host protein were diluted to 1.0 μg / ml with carbonate buffer, respectively, and coated on 96-well enzyme-labeled plates at 4°C overnight. 0.01M phosphate buffer (PBST) with a final concentration of 0.05% Tween 20 was added for washing 3-5 times, 150 μl of PBST containing 3-6% BSA was added to each well for blocking at 37°C for 1-2 hours, and the blocking solution was discarded and dried. The test monoclonal antibody and negative control (sample dilution) were serially diluted by 10 times according to the 10-fold gradient method to 10 8 -2 -8 The 10 5 dilution samples were added to the above 96-well plates, 100 μl was added to each well, 37°C incubation for 1 hour, 300 μl of PBST was added to each well, and washing was performed 2-5 times, 1:5000 diluted goat anti-mouse IgG-HRP was added to each well, 100 μl, 37°C incubation for 45 minutes, and the plate was washed with PBST 2-5 times, dried, and substrate color developing solution was added to develop color at room temperature for 10-15 minutes. The reaction was terminated with 1M H2SO4, and the reading was performed at 450nm on a microplate reader. The negative and positive critical value judgment standard was that the value should be ≥ the mean value of the negative control x 2.1. The results showed that the monoclonal antibody 24G9 was specifically combined with CV-A16 antigen, the binding antibody titer was 1.28*10 50 , and it was not combined with other types and Vero cell host protein, indicating that it had good specificity.

[0101] Neutralizing antibody titer assay: the purified monoclonal antibody 24G9 (previously diluted to 10 μg / ml) was diluted 1:8, added to a 96-well cell culture plate, and serially diluted by 2 times, 0.05 ml / well. 100 CCID 5 (50% cell infection dose) of CV-A16 / CV-A6 / CV-A10 / EV-A71 virus suspension was neutralized at 37°C for 2 hours; 1-2*10 50 cells / ml of RD cell suspension was added, 0.1 ml / well, and incubated in a 35±0.5°C, 5% CO2 incubator for 7 days. The highest dilution that could inhibit 50% of the cytopathic effect was defined as the neutralizing antibody titer, and the reciprocal of the dilution factor was used for expression. Virus back titration was set for each test, and the back titration result was 32-320 CCID 50 / well, which was considered to be true. Neutralizing antibody positive was defined as neutralizing titer ≥8, and negative was <8. The geometric mean titer (GMT) of the negative antibody was calculated as 4. The results are shown in Table 1. Figure 3As shown, the results showed that the neutralizing antibody titer of monoclonal antibody 24G9 against CV-A16 strain was 1:8192 (0.0012 μg / ml), and the neutralizing antibody titer of monoclonal antibody 24G9 against EV-A71, CV-A6, CV-A10 strains was <1:8, indicating that the monoclonal antibody had high in vitro neutralization activity and did not cross-react with other types of enterovirus.

[0102] Subtype identification: The purified antibody 24G9 was diluted to 200 ng / ml with PBST, 50 μl / well was added to a pre-coated enzyme-labeled plate (manufacturer: Proteintech), 1x goat anti-mouse IgM+IgG-HRP was added to the sample well, 50 μl / well, gently mixed, and incubated at room temperature for 1 hour. Discard the liquid in the well, wash the plate with PBST for 3 times, and pat dry. Add color developing solution, 100 μl / well. Incubate at room temperature for 10-20 min. Add 100 μl / well of stop solution to stop the reaction. Read OD450nm by an enzyme-labeled instrument. The OD value of the deepest well corresponds to the corresponding subtype. The subtype determination results of monoclonal antibody 24G9 are shown in Table 1, the heavy chain is IgG3, and the light chain is Kappa.

[0103] Table 1 Subtype identification results

[0104] Subtype classification IgGl IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda Absorbance 450 nm 0.0839 0.0613 0.0564 0.0528 1.8539 0.0561 0.4219 0.0557

[0105] Immunofluorescence staining: Vero cells infected with CV-A16 virus for 24-48 hours, discard the culture solution, wash with PBS for 3 times, and then fix with pre-cooled 85% acetone for 15-20 min. The fixed cells were blocked with PBS containing 3% BSA at 37°C for 1 hour. The monoclonal antibody 24G9 was diluted to a final concentration of 0.1 μg / ml with PBS containing 3% BSA, and then incubated with the cells at 37°C for 1 hour. Wash with PBS for 3 times, dilute the FITC-labeled anti-mouse IgG (Solebao, China) 100 times as a detection secondary antibody, and incubate at 37°C for 1 hour. Wash with PBS for 3 times. Observed by an immunofluorescence microscope (Olympus, China).

[0106] As shown in the results, monoclonal antibody 24G9 can detect CV-A16 virus infected cells by immunofluorescence staining method. Figure 4

[0107] ​The competitive inhibition detection of human serum (healthy human serum, human natural mixed infection serum, and human natural infection serum 1, wherein the human natural mixed infection serum is obtained by mixing 16 human natural infection sera from flow investigation, and the 16 human natural infection sera from flow investigation are all specific infection CV-A16 serum samples detected by serology): the antigen of CV-A16 is diluted to 0.01 μg / ml and coated on a 96-well enzyme-labeled plate, the human serum is diluted with a sample diluent from 1:4, continuously diluted by 2 times, 100 μL / well, and incubated at 37°C for 1 h; 24G9-HRP (HRP-labeled monoclonal antibody 24G9) is diluted by 1:200 with an antibody diluent, 100 μL / well, and incubated at 37°C for 1 h; the average absorbance value of the control hole is taken as B0, and the average absorbance value of the sample group is taken as B1, and the inhibition rate % = (B0-B1) / B0*100. The inhibition rate is taken as the vertical coordinate, and the serum dilution degree (log2 as the base) is taken as the horizontal coordinate, and the reaction curve (R 2 The inhibition rates are 0.9921, 0.9885, and 0.9169 in turn.

[0108] The results are shown in Table 1. Figure 5 As shown in Table 1, the healthy human serum has no obvious competitive inhibition with the monoclonal antibody 24G9, and the human natural mixed infection serum and the human natural infection serum have obvious competitive inhibition with the monoclonal antibody 24G9, and the inhibition rate of the human natural mixed infection serum reaches more than 96%. It is proved that the epitope corresponding to the monoclonal antibody 24G9 occupies an important position in the immune response caused by human infection.

[0109] The gene sequence of the monoclonal antibody is isolated and identified: total RNA is extracted from the well-grown hybridoma cell strain by using Trizol reagent, cDNA is synthesized by reverse transcription, and then the light chain and heavy chain variable region genes are amplified by PCR, and the amino acid sequence is determined. The results show that the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2, and CDR3 of the monoclonal antibody 24G9 are shown in SEQ ID NO. 1-3, the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 4-6; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 11; the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 12; the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO. 13, the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO. 14; the nucleotide sequence of the gene encoding the heavy chain is shown in SEQ ID NO. 15, and the nucleotide sequence of the gene encoding the light chain is shown in SEQ ID NO. 16.

[0110] Example 4 Detection of specific binding ability of CV-A16 virus particle antigen

[0111] Sandwich ELISA experiment: Anti-CV-A16 rabbit polyclonal antibody was diluted with coating solution carbonate buffer (CBS pH 9.6) (1:1000, 1:5000, 1:10000, 1:20000), added to the enzyme labeling plate (Corning, detachable), 100 μl per well, and coated overnight at 2-8°C; washed 3 times with PBST and patted dry; blocked with PBST containing 3% BSA, 150 μl per well, incubated at 37°C for 2h; washed 3 times with PBST and patted dry; diluted the antigen standard (a mixture of hollow virus particles and solid virus particles, prepared and sold by the China Food and Drug Inspection Institute as a national antigen standard (2016 "Guoshengbiaozi" 0031), batch number 201507001) to 200U / ml with sample diluent (PBS solution containing 0.5% BSA) As a positive control, the sample diluent was used as a negative control. 100 μl of the sample diluent was added to each well and incubated at 37°C for 2 h. The cells were washed three times with PBST and patted dry. Monoclonal antibody 24G9 was diluted in antibody diluent (PBST containing 10% FBS) to the appropriate concentrations (1:1000, 1:5000, 1:10000, 1:20000) and incubated at 37°C for 1 h. The cells were washed three times with PBST and patted dry. Goat anti-mouse HRP was diluted in antibody diluent to 1:8000 and incubated at 37°C for 30-60 min. The cells were washed four times with PBST and patted dry. 100 μl of TMB substrate developer was added to each well and developed at room temperature in the dark for 15 min. 50 μl of 1 mol / L H₂SO₄ was added to each well. The concentrations were read at 450 nm / 630 nm. The results showed that the P / N value was the largest when the coated polyclonal antibody was diluted 1:10000 and the monoclonal antibody 24G9 was diluted 1:5000 as the detection antibody, indicating that the monoclonal antibody 24G9 can be used as a detection antibody for the detection of a mixture of hollow virus particles and solid virus particles (in the practice of virus liquid production, the virus particles produced are also a mixture of hollow and solid virus particles).

[0112] Example 5 Horseradish peroxidase labeled monoclonal antibody

[0113] HRP 5mg was dissolved in 0.2mol / L, pH 5.6 acetate buffer 0.5ml; added freshly prepared 0.1mol / L NaIO40.25ml, at this time the solution changed from original brown to dark green, placed at 4°C for 30min, at this time the solution changed from original brown to dark green; added 2.5% ethylene glycol 0.5ml, gently stirred at room temperature for 1h, terminated the reaction; added 10mg monoclonal antibody 24G9, adjusted pH value to 9.0 with 1.0mol / L, pH 9.5 CBS; mixed well, placed at 4°C overnight; added sodium borohydride solution 0.1ml, mixed well, placed at 4°C for 3h; dialyzed overnight at 4°C with 0.15mol / L, pH 7.4 PBS solution, changed 3 times; centrifuged at 3000r / min for 30min, removed the precipitate; collected the supernatant, which was the enzyme-labeled antibody 24G9. Added equal volume of 60% glycerol, small amount of packaging, stored at below -20°C.

[0114] Determination of the titer of enzyme-labeled antibody: CV-A16 antigen was first diluted to about 10μg / ml with 0.05mol / L pH 9.6 coating buffer, 100μl was added to polystyrene plate wells, and incubated at 4°C overnight, washed 3 times with PBST. The plate was blocked with 3% BSA-PBST, 150μl / well, incubated at 37°C for 2h, washed 3 times with PBST. The enzyme-labeled antibody was diluted from 1:100 with 0.5% BSA-PBST, 2-fold serial dilution to 12800, added to the reaction wells, 100μl per well, incubated at 37°C for 1h, then washed. Then added color developing solution, 100μl per well, room temperature for 10min in the dark. The reaction was terminated with 1mol / L H2SO4, 50μl per well.

[0115] The OD450nm / 630nm value of each well was read by ELISA enzyme marker, and the OD(450nm-630nm) was taken as the vertical coordinate, and the enzyme-labeled antibody dilution was taken as the horizontal coordinate, to draw the titration curve. The enzyme-labeled antibody dilution when the OD value was about 1.0 and the curve slope was the largest was the titer of the antibody of the marker. The results are shown in Table 2 and Figure 6 , which shows that the titer of enzyme-labeled antibody 24G9 was 51200.

[0116] Table 2 Absorbance of enzyme-labeled antibody reaction

[0117] Dilution factor Absorbance 1 Absorbance 2 Absorbance mean 3200 3.816 3.793 3.804 6400 3.522 3.437 3.480 12800 2.951 2.877 2.914 25600 2.019 1.893 1.956 51200 1.133 1.024 1.078 102400 0.590 0.544 0.567 204800 0.318 0.294 0.306 409600 0.156 0.144 0.150 819200 0.074 0.062 0.068 1638400 0.025 0.021 0.023

[0118] Establishment of antigen detection method

[0119] The rabbit polyclonal antibody was used as the coating antibody, and the 24G9 monoclonal antibody was used as the detection antibody to establish a double antibody sandwich enzyme-linked immunoassay method for detecting the CV-A16 antigen. The chessboard method was used for the test, and the specific method was as follows: the rabbit polyclonal antibody was diluted to 1.6 μg / ml, 0.32 μg / ml, 0.16 μg / ml, 0.08 μg / ml with a carbonate buffer (pH 9.6), and was coated on a 96-well enzyme-labeled plate at 100 μl / well and 37°C for 2 hours. The plate was washed with PBST for 3 times, and the non-specific binding sites were blocked with a blocking solution (0.01M PBS containing 3% BSA and 0.5‰ Tween 20) at 150 μl / well and 37°C for 2 hours. The CV-A16 vaccine antigen was diluted to 100 U / ml with PBS (containing 0.5% BSA), and was added to the enzyme-labeled plate at 100 μl / well, and the sample diluent was used as the negative control, and a blank control was set at the same time, 2 wells for each, and 37°C for 2 hours. The plate was washed with PBST for 4 times, and the HRP-labeled 24G9 monoclonal antibody (1:1000, 1:2000, 1:5000, 1:10000) was added and incubated at 37°C for 30-45 min. The plate was washed with PBST for 5 times, 100 μl of TMB color developing solution was added to each well, and the color was developed at room temperature for 15 min in the dark, 50 μl of the termination solution (1M sulfuric acid solution) was added to each well to terminate the reaction, and the value was read at 450 / 630 nm on an enzyme-labeled instrument. The detection results are shown in Table 3. When the P / N value was the highest, the coating antibody was coated at 0.08 μg / ml, and the enzyme-labeled antibody was diluted at 1:10000, and this combination was the best. The double antibody sandwich method with 24G9 as the detection antibody can be used for the detection of the CV-A16 antigen.

[0120] Table 3 Chessboard method

[0121]

[0122] Note: P represents the mean value of the sample to be tested, and N represents the mean value of the negative control.

[0123] Example 7 Evaluation of the antigen detection method

[0124] Linear range: According to the determined antigen detection method, the national antigen standard was diluted by 2 times continuously for 8 dilution degrees (400 U / ml, 200 U / ml, 100 U / ml, 50 U / ml, 25 U / ml, 12.5 U / ml, 6.25 U / ml, 3.125 U / ml) from 400 U / ml, and each dilution degree was made in duplicate. Independent determination was made for 6 times. The logarithm of the content of the standard was taken as the abscissa, and the logarithm of the absorbance value was taken as the ordinate, a standard curve was drawn, and the consistency of the results of 6 times was investigated. The 2.1* negative mean value was used as the judgment standard of the positive and negative. 2

[0125] The R 2 of the results of 6 tests was greater than 0.98, which is shown in​Figure 7 , indicating that the method has good linearity and parallelism, and the standard has good linearity within the range of 400 to 3.125 U / ml.

[0126] Specificity evaluation: Referring to the method of Example 6, EV-A71, CV-A6, and CV-A10 antigens, which belong to the same Picornaviridae family as CV-A16, and Vero cell host proteins used in production were diluted to 1000 ng / mL as test samples (calculated by protein content), as well as poliovirus (PV) antigen and other components that may be present in the CV-A16 antigen sample, M199, DMEM, PEG6000, 58% sucrose, and 10 mmol / L PBST (1 M NaCl, 0.1% Tween 80) as test samples (abbreviated as S). CV-A16 antigen was used as a positive control, the sample dilution was used as a negative control, and 2.1 times the negative control was used as the CUT-OFF value (abbreviated as CO) to verify the specificity of the antigen detection system for enterovirus detection.

[0127] The results are as follows Figure 8 As shown, the antigen evaluation system has good specificity and does not react to other types of enterovirus. This shows that the antigen evaluation system provided by the present invention has exclusive specificity for CV-A16 virus detection.

[0128] Accuracy: The national standard (2000 U / ml) was diluted to 150 U / ml, 50 U / ml, and 10 U / ml as the test samples for the high, medium, and low concentrations. The experiment was repeated six times by a single person according to Example 6. The recoveries and confidence intervals for the three concentrations were calculated. The recovery was calculated as measured value / theoretical value × 100%. The results showed that the recoveries ranged from 80% to 120% (Table 4), which is within the acceptable range.

[0129] Table 4 Accuracy-antigen recovery %

[0130] Number of experiments 150 U / ml 50 U / ml 10 U / ml 1 96.7 108.0 90.0 2 97.0 106.0 80.0 3 80.7 120.0 100.0 4 100.7 110.0 90.0 5 96.0 102.0 110.0 6 89.3 90.0 90.0 Mean 93.4 106.0 93.3 95% confidence interval 85.8～101 95.6～116 92.3～102.9

[0131] Precision: Three independent assays were performed three times at different times using 150 U / ml, 50 U / ml, and 10 U / ml national antigen standards as test samples, according to Example 6. The CV values ​​were calculated for each assay. The results are shown in Tables 5 and 6. The results show that the CV values ​​for the three assays performed by the three assays were between 5.43% and 10.44%, while the CV values ​​for the three assays performed by assayer 1 were between 5.02% and 6.66%, indicating that the assay has good repeatability and intermediate precision.

[0132] Table 5 Intermediate precision

[0133]

[0134] Table 6 repeatability

[0135]

[0136] Evaluation of the antigen detection kit on the binding ability of empty solid particles: according to the determined antigen detection method, the empty virus particles and solid virus particles are respectively diluted according to a certain concentration series, and under the condition of reaching the same OD value, the inverse ratio of the concentration of empty and solid virus proteins is the ratio of the reaction ability of the antigen detection kit to empty virus particles and solid virus particles.

[0137] The results show that the reaction ability of empty and solid particles is 8:1 (Table 7). The antigen detection kit can well detect empty and solid particles.

[0138] Table 7 Comparison of reaction ability of empty and solid particles

[0139]

[0140] Example 8 Monoclonal antibody 24G9 in vivo treatment of CV-A16 infection experiment

[0141] The 1-day-old BALB / c mice were infected with a lethal dose (448 CCID 50 / mouse) of CV-A16 strain numbered R00880662 (preserved number CGMCC No. 19534, which has been disclosed in patent CN113564132B), including 8 groups of virus control groups, 0.5h after the experiment group was injected with 10.0 μg / g, 2.0 μg / g, 0.4 μg / g, 0.08 μg / g, 0.016 μg / g, 0.0032 μg / g of monoclonal antibody 24G9, and the blank control group was injected with the same dose of MEM. The survival state of the mice was observed and recorded for 21 consecutive days, and the survival curve was drawn.

[0142] Among them, all the mice in the MEM control group survived, and 100% of the mice in the virus control group died 9 days after the infection. The results show that Figure 9 ): the mice in the 10 μg group can all survive and do not show clinical symptoms such as emaciation and limb paralysis; 83% of the mice in the 2 μg group survived, 50% of the mice in the 0.4 μg and 0.08 μg groups survived, and 33% of the mice in the remaining groups survived. It is shown that the monoclonal antibody 24G9 can be used as a candidate drug for treating CV-A16 infection.

[0143] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An anti-Coxsackievirus A16 antibody or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs. 1-3, and the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody is a monoclonal antibody; the antigen-binding fragment is Fab, Fab', F(ab')2, Fd, Fv, dAb or single-chain antibody.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule according to claim 4, and the biological material is an expression cassette, a vector or a host cell.

6. An antibody conjugate, characterized in that The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 is coupled to a label, wherein the label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The method comprises: culturing a host cell capable of expressing the antibody or the antigen-binding fragment thereof, and obtaining the antibody or the antigen-binding fragment thereof by separation.

8. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, or the antibody conjugate according to claim 6: (1) Use in the preparation of a product for detecting the presence or level of Coxsackievirus A16 in a sample; (2) Use in the preparation of products for diagnosing Coxsackievirus A16 infection or diseases caused by Coxsackievirus A16 infection; (3) Use in detecting the presence or level of Coxsackievirus A16 in a sample for non-diagnostic and non-therapeutic purposes; (4) Application in detecting the antigenicity or immunogenicity of Coxsackievirus A16 vaccine; (5) Application in quality control of Coxsackievirus A16 vaccine production; (6) Use in the preparation of products for detecting the specificity or content of Coxsackievirus A16 antigen; (7) Use in the preparation of a product for neutralizing the virulence of Coxsackievirus A16 in a sample; (8) Use in the preparation of drugs for preventing or treating Coxsackievirus A16 infection or diseases caused by Coxsackievirus A16 infection.

9. A kit, characterized in that The kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 6.

10. A drug, characterized in that The drug comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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