CN110981955B - Monoclonal antibody of coxsackievirus A10 type hollow virus and application thereof - Google Patents
Monoclonal antibody of coxsackievirus A10 type hollow virus and application thereof Download PDFInfo
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- CN110981955B CN110981955B CN201911380621.6A CN201911380621A CN110981955B CN 110981955 B CN110981955 B CN 110981955B CN 201911380621 A CN201911380621 A CN 201911380621A CN 110981955 B CN110981955 B CN 110981955B
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Abstract
The invention discloses a monoclonal antibody capable of reacting with coxsackievirus A10 type hollow virus and a detection kit. The invention adopts the purified CA10 virus liquid to immunize mice to prepare monoclonal antibodies. The invention also discloses application of the monoclonal antibody in detecting CA10 virus or diagnosing hand-foot-and-mouth disease. The monoclonal antibody has wide application in the preparation of a rapid detection kit and the development and research of vaccines.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to a monoclonal antibody of a coxsackievirus A10 type hollow virus and application thereof.
Background
The hand-foot-and-mouth disease is an acute infectious disease caused by various enterovirus infections, is epidemic in summer, and is high in children before school age, and adults can be indirect infectious sources. The hand-foot-and-mouth disease is clinically mainly manifested as skin rash of the oral cavity, hands and feet, and can be complicated with meningitis, encephalitis, pulmonary edema, circulatory failure and other serious deaths. Enterovirus 71 (enterovirus 71, EV71) and coxsackievirus A16(coxsackievirus, CA16) are the most common pathogens causing hand-foot-and-mouth disease in the continental area of China. However, with the progress of detection techniques and virus typing methods, it has been found in recent years that the prevalence rates of coxsackie virus a6(coxsackievirus, CA6) and coxsackie virus a10(coxsackievirus, CA10) in enteroviruses tend to increase year by year and become the main prevalent serotypes in some regions. Aiming at the epidemic trend of virus serotypes in recent years, a plurality of research institutions and enterprises at home and abroad are all engaged in developing vaccines or medicines capable of effectively preventing the hand-foot-and-mouth diseases caused by CA6 or CA 10.
In order to provide the fastest possible clinical protocols, rapid detection methods are generally required, and ELISA rapid detection kits are the most commonly used methods, which can detect the serotype of the virus infected by a patient in the shortest time. The screening of a monoclonal antibody with type specificity is a precondition for establishing a detection kit. In addition, in the vaccine research process, the antigen content in the vaccine is a key index for guiding process research and evaluating in vitro effectiveness. The most key technology for establishing the antigen evaluation method is to screen a proper monoclonal antibody, and then an effective ELISA evaluation system can be established by using the monoclonal antibody.
Coxsackievirus A6(coxsackievirus, CA6) and coxsackievirus A10(coxsackievirus, CA10) are enteroviruses, which easily form two different structural states when cultured in vitro, one is a complete solid virus particle containing nucleic acid, and the other is a hollow virus particle without nucleic acid, and usually the virus particles in the two states coexist. The solid virus particles and the hollow virus particles are both immunogenic, but the solid virus particles are obviously stronger than the hollow virus particles, which is shown by literature records and multiple experimental researches. If the virus stock solution contains hollow virus particles in a certain proportion, the total protein content of the stock solution can be effectively improved, and the virus stock solution has important significance on the stability of the virus stock solution and finished vaccines. Therefore, in the process research and the preparation research of vaccines, the ratio of solid virus particles to hollow virus particles in the product can be determined, so that the important significance is realized on the quality evaluation of the product. Establishing an antigen evaluation system capable of detecting solid virus particles and an antigen evaluation system capable of detecting hollow virus particles respectively has important significance for monitoring virus stock solution and controlling the quality of finished vaccines.
An antigen evaluation system (including an ELISA rapid detection kit) usually adopts a double-antibody sandwich method, such as a polyclonal antibody (short for polyclonal antibody) -polyclonal antibody, a polyclonal antibody-monoclonal antibody (short for monoclonal antibody), a monoclonal antibody-polyclonal antibody, a monoclonal antibody-monoclonal antibody, and the use of polyclonal antibody is easy to cause cross binding among different types of enteroviruses and has weak detection specificity on the viruses; the monoclonal antibody is easy to be used, the binding capacity of the antigen epitope is insufficient, and the real content of the antigen cannot be objectively reflected.
There is no report in the literature on an antigen evaluation system capable of detecting mainly CA10 hollow virus particles. In the existing process research, the proportion of hollow viruses and solid viruses in the virus liquid is determined mainly by means of electron microscope observation, the electron microscope observation method has high requirements on the concentration of a sample and the components of a buffer system, the test is long in time consumption, the requirements on instruments and equipment in a laboratory are high, and the method has certain limitations.
Disclosure of Invention
In order to solve the technical problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides a monoclonal antibody against CA10 virus, said monoclonal antibody having heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 5, heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 6, heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 7, and light chain complementarity determining region CDR1 shown in SEQ ID NO. 13, light chain complementarity determining region CDR2 shown in SEQ ID NO. 14, light chain complementarity determining region CDR3 shown in SEQ ID NO. 15; preferably, the monoclonal antibody has the heavy chain of the amino acid sequence shown in SEQ ID NO. 8; and/or, preferably, the monoclonal antibody has the amino acid sequence shown in SEQ ID NO. 16 of the light chain;
preferably, the CA10 virus is a CA10 empty virus.
In a second aspect, the present invention provides a polynucleotide sequence encoding the monoclonal antibody against CA10 virus, the polynucleotide sequence having the sequence encoding heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 1, the sequence encoding heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 2, the sequence encoding heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 3, and the sequence encoding light chain complementarity determining region CDR1 shown in SEQ ID NO. 9, the sequence encoding light chain complementarity determining region CDR2 shown in SEQ ID NO. 10, the sequence encoding light chain complementarity determining region CDR3 shown in SEQ ID NO. 11; preferably, the polynucleotide sequence has a nucleotide sequence shown as SEQ ID NO. 4; and/or, preferably, the polynucleotide sequence has the nucleotide sequence shown in SEQ ID NO. 12;
preferably, the CA10 virus is a CA10 empty virus.
In a third aspect, the present invention provides a kit for detecting the CA10 virus, said kit comprising said monoclonal antibody or a monoclonal antibody encoded by said polynucleotide sequence; preferably, the kit further comprises a polyclonal antibody; more preferably, the polyclonal antibody is a CA10 rabbit polyclonal antibody;
preferably, the CA10 virus is a CA10 empty virus.
In a fourth aspect, the invention provides a kit for diagnosing hand-foot-and-mouth disease, wherein the kit comprises the monoclonal antibody or the monoclonal antibody coded by the polynucleotide sequence; preferably, the kit further comprises a polyclonal antibody; more preferably, the polyclonal antibody is a CA10 rabbit polyclonal antibody;
preferably, the CA10 virus is a CA10 empty virus.
In a fifth aspect, the invention provides an application of the monoclonal antibody against the CA10 virus or the monoclonal antibody coded by the polynucleotide sequence in preparing a kit for detecting the CA10 virus or diagnosing hand-foot-and-mouth disease;
preferably, the CA10 virus is a CA10 empty virus.
In a sixth aspect, the invention provides the use of the monoclonal antibody against CA10 virus or the monoclonal antibody encoded by the polynucleotide sequence for quality control of the production of a vaccine comprising CA10 virus;
preferably, the CA10 virus is a CA10 empty virus.
In a seventh aspect, the invention provides a method of quality control of production of a vaccine comprising CA10 virus, the method comprising the step of detecting CA10 virus with said monoclonal antibody against CA10 virus or a monoclonal antibody encoded by said polynucleotide sequence;
preferably, the CA10 virus is a CA10 empty virus.
In an eighth aspect, the present invention provides a medicament for treating or preventing a disease caused by infection with CA10 virus, said medicament comprising said monoclonal antibody against CA10 virus or said monoclonal antibody encoded by said polynucleotide sequence;
preferably, the CA10 virus is a CA10 empty virus.
In a ninth aspect, the invention provides an application of the monoclonal antibody against the CA10 virus or the monoclonal antibody coded by the polynucleotide sequence in detecting the content of the CA10 virus in the preparation process of a hand-foot-and-mouth vaccine or a vaccine finished product.
Preferably, the application is used for detecting the content of CA10 empty virus in the preparation process of the hand-foot-and-mouth vaccine or in the vaccine finished product.
The invention has the beneficial effects that:
the invention provides an antigen evaluation system for mainly detecting CA10 hollow virus particles. The antigen evaluation system prepared by the monoclonal antibody provided by the invention can be used for rapidly detecting the hollow virus, and the binding capacity of the hollow virus to the solid virus is weak. The proportion of the solid virus particles is an important focused index in a vaccine research process, and is positively correlated with the effectiveness of a product, the immunogenicity of the hollow virus particles is not as good as that of the solid virus particles, but the stock solution or a finished product contains a certain proportion of the hollow virus particles, so that the stability and the immunogenicity of the hollow virus particles can be enhanced. In the production stage and the purification stage of the virus stock solution, the proportion of the empty and solid viruses in the product in each step is monitored in real time, and the method has guiding significance for selecting the harvesting time of virus fermentation and combining virus solutions after ultracentrifugation. The hollow antigen evaluation system provided by the invention is combined with a solid antigen evaluation system for use, and has important significance on the production process research of vaccines.
Of course, the antigen evaluation system can also be applied to the preparation of a rapid detection kit and the research and development of vaccines.
Drawings
FIG. 1 is a photograph of a centrifuge tube after sucrose density gradient centrifugation in example 1;
FIG. 2 is an electron micrograph of solid and hollow virus particles identified in example 1, wherein: the left figure is a 22 # tube solid virus particle, and the right figure is a 19 # tube hollow virus particle;
FIG. 3 is the SDS-PAGE electrophoresis of the purified mAb of example 3, in which: 2-dilution of the sample before purification, 3-ascites before purification, 4-purification of the eluate (heavy chain + light chain);
FIG. 4 is a linear relationship diagram of the antigen evaluation system of example 5;
FIG. 5 is a graph showing the evaluation of the antigen evaluation system of example 7 for a sample in the fractionation process.
Description of sequence listing
Note: the underlined font part is the CDR region and the bolded font part is the CH1 end primer.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 preparation of solid and hollow viral particles of CA10
And (3) culturing the virus: the CA10 virus strain is adopted, and the cells for culture are Vero cells (from WHO world health organization). Culturing cells by adopting a mode of fermentation tank microcarrier, inoculating the virus into a fermentation tank according to the MOI of 0.0001-0.001, culturing at the temperature of 36.0 +/-0.5 ℃, culturing the virus for 1-5 days, then harvesting to obtain CA10 virus liquid, and clarifying and ultrafiltering for primary purification by adopting a membrane package with the KD of 100-300 KD.
Sucrose density gradient centrifugation yields viral particles of different nature: and (3) performing air-solid separation and impurity protein removal on the CA10 virus liquid after primary purification by adopting a sucrose density gradient centrifugation method. The sucrose gradient is 15% -60%, and the centrifugation is carried out for 3-15 hours at 100000 revolutions. And (3) centrifugally separating a hollow virus band and a solid virus band by sucrose density gradient, respectively taking out the two virus bands, and identifying the hollow and solid separation result by using an electric mirror.
The photograph of the centrifuge tube after sucrose density gradient centrifugation is shown in FIG. 1, from which the separated hollow virus bands and solid virus bands can be seen. The electron microscope comparison chart of solid virus particles and hollow virus particles identified by electron microscope is shown in FIG. 2.
Example 2 preparation of a monoclonal antibody to CA10 hollow Virus
Preparing hybridoma cell strains: a CA10 hollow virus purified solution (a preparation method of the hollow virus purified solution is that a Vero cell is used for culturing a CA10 virus, the virus solution is obtained, then the virus solution is clarified, ultrafiltered, primarily purified and concentrated, and then is centrifuged by sucrose gradient density to obtain a solid virus particle tube and a hollow virus particle tube, and the hollow virus particle tubes are collected and then are desugared to obtain the CA10 hollow virus purified solution) for immunizing BALB/c mice, wherein 5 mice are immunized per type; 4 needles in total for subcutaneous multiple immunizations on the back at 0, 2, 4, 6 weeks; immunization dose: 0.2 ml/needle/stick; adjuvant: the 1 st needle is Freund's complete adjuvant, the 2 nd and 3 rd needles are Freund's incomplete adjuvant, and the 4 th needle is not added with adjuvant; blood sampling detection: the blood is collected 1 week after the 3 rd injection of immunity and the titer of the indirect enzyme-linked immunosorbent assay is detected, and the titer of the antibody reaches 104The above mice were injected intraperitoneally with the 5 th needleAnd (4) boosting immunity.
Cell fusion: 3 days after the intraperitoneal injection for boosting immunity, the mice are killed, the spleen is taken out for fusion, after two times of monoclonality, positive hybridoma cell strains (the OD450 value of cell supernatant is more than 1.0) are obtained, and then the mice are immunized to prepare ascites.
Taking out the frozen CA10 hollow virus mouse monoclonal hybridoma cell strain from liquid nitrogen for recovery and expanded culture, 106Extracting total nucleic acid in the above amount, PCR amplifying the heavy chain and light chain sequence of the monoclonal antibody by Beijing Liu-Hua Dagenescience and technology Limited, sequencing, determining the corresponding amino acid sequence by nucleotide sequence, and referring the determined polynucleotide sequence and amino acid sequence to the sequence table.
Example 3 purification of hollow virus monoclonal antibody CA10
Antibody purification: and (3) centrifuging the ascites prepared by immunizing the mouse in the example 2 at the temperature of 2-8 ℃ and at the speed of 4000-8000 r/min for 5-15 minutes, taking the supernatant, filtering the supernatant by qualitative filter paper, filtering the supernatant by a 0.45 mu m filter membrane, and performing affinity chromatography to obtain the purified monoclonal antibody. After purification, the protein content was determined to be 32563. mu.g/ml. And (4) carrying out purity detection and titer determination on the purified monoclonal antibody.
(1) Detection of purity of monoclonal antibody
And (3) performing SDS-PAGE electrophoresis on the purified monoclonal antibody, detecting the proportion of IgG heavy chain and light chain, and analyzing the purity of the monoclonal antibody by using a gel imaging scanner, wherein the SDS-PAGE electrophoresis chart after the purification of the monoclonal antibody is shown in figure 3, and two groups are respectively tested, wherein 2 in the figure is a diluted sample before purification, 3 is ascites before purification, and 4 is a purification eluent (heavy chain + light chain).
(2) Determination of the potency of the monoclonal antibody
Pre-coating: the CA10 purified solution is diluted to 0.5-2.0 mu g/ml by 0.01M phosphate buffer solution, and a 96-hole enzyme label plate is coated at 4 ℃ overnight or 37 ℃ for 2 hours. And (3) adding 0.01M phosphate buffer solution with the final concentration of 0.05% Tween 20, washing for 2-5 times, adding 0.01M phosphate buffer solution containing 5-20% calf serum, sealing for 1-2 hours at 37 ℃, and throwing off the sealing solution and clapping to remove residual sealing solution in the holes when in use.
And (3) measuring the titer: the serum to be detected and the negative serum control are 10 timesGradient method for serial gradient dilution to 108Adding 10 times of2To 108Adding 100 μ l of diluted sample into the above 96-well plate, incubating at 37 deg.C for 0.5-2 hr, washing with 0.01M PBST20 lotion for 2-5 times, incubating at 37 deg.C for 0.5-2 hr with anti-mouse IgG HRP (commercially available from KPL manufacturer), washing for 2-5 times, drying, adding chromogenic substrate, developing at 37 deg.C for 8-15 min, and 2M H2SO4The reaction was terminated and read at a wavelength of 450 nm. The standard is as follows: under the same dilution times, the OD450 value of the sample is more than or equal to negative control multiplied by 2.1, if the light absorption value of the negative control is less than 0.05, the positive standard is judged by calculating with 0.05. The titer is 10 by adopting an ELISA indirect method7。
The proportion of Marker (Marker) protein bands in FIG. 3 to total protein in the lanes is shown in the following table.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |
Marker substance | 0.0955 | 0.0856 | 0.1110 | 0.1481 | 0.0996 | 0.2233 | 0.0888 | 0.0755 | 0.0465 | 0.0261 |
The proportion of the heavy chain and the proportion of the light chain of the purified eluent obtained by detection are respectively as follows: the heavy chain is 0.5769 and the light chain is 0.4231. From the results, the sum of the ratio of the heavy chain and the light chain in the purified monoclonal antibody eluent is 100%, which shows that the purification effect is good and no impurity protein exists.
Example 4 Standard enzyme of the hollow Virus monoclonal antibody CA10
Placing the purified monoclonal antibody into a dialysis bag, dialyzing for 2-8 hours in a 0.05M carbonate buffer solution system, and changing the dialysate once every 1-2 hours; HRP was activated with sodium periodate and 20% ethylene glycol was used to stop the activation. Adding the activated HRP into the antibody, and continuously dialyzing overnight; taking out the antibody to obtain a HRP-labeled CA10 hollow virus monoclonal antibody, and adding sodium borohydride for reduction; ammonium sulfate precipitation, re-dissolving with 0.01MPBS, and storing at-20 deg.C or below.
Example 5 antigen evaluation System matching
Diluting CA10 rabbit polyclonal antibody (prepared by Beijing Kexing biological products Co., Ltd., Beijing Kexing for short) with carbonate buffer solution according to a certain proportion, and coating 96-hole enzyme label plate at 4 deg.C overnight or 37 deg.C for 2 hours. And (3) adding 0.01M phosphate buffer solution with the final concentration of 0.05% Tween 20, washing for 2-5 times, adding 0.01M phosphate buffer solution containing 5-20% calf serum, sealing for 1-2 hours at 37 ℃, and throwing off the sealing solution and clapping to remove residual sealing solution in the holes when in use. When the technical scheme related by the invention is used for preparing the kit, an enzyme-labeled dry plate can be prepared in advance and cultured at the temperature of 2-8 ℃.
Antigen dilution: carrying out serial gradient dilution on CA10 virus liquid according to a certain concentration, sequentially adding 40U/ml, 20U/ml, 10U/ml, 5U/ml and 2.5U/ml into the 96-well plate, adding 100 mu l of each well, incubating for 0.5-2 hours at 37 ℃, washing for 2-5 times by 0.01M PBST20 washing liquid, adding the HRP-labeled CA10 hollow virus monoclonal antibody prepared in example 4, incubating for 0.5-2 hours at 37 ℃, washing for 2-5 times, patting dry, adding a chromogenic substrate, developing for 8-15 minutes at 37 ℃, and developing for 2M H minutes2SO4The reading is terminated at the wavelength of 450-630 nm. The sensitivity and linear relationship of the antigen evaluation system were examined.
The detection sensitivity of the antigen evaluation system to the CA10 virus is 5U/ml, and the linear correlation R2The result is more than or equal to 0.98, the result is shown in table 1, and the linear relation graph of the antigen evaluation system is shown in fig. 4.
TABLE 1 results of sensitivity detection
Virus antigen (U/ml) | OD value 1 | OD value 2 | OD mean value |
Blank hole | 0.072 | 0.064 | 0.068 |
2.5 | 0.142 | 0.135 | 0.139 |
5 | 0.239 | 0.218 | 0.229 |
10 | 0.451 | 0.460 | 0.456 |
20 | 0.893 | 0.905 | 0.899 |
40 | 1.751 | 1.764 | 1.758 |
Example 6 evaluation of binding Capacity of antigen evaluation System to solid Virus particles or hollow Virus particles
Referring to the method in example 5, CA10 solid virus particles (from beijing kakking) and CA10 hollow virus particles (from beijing kakking) were serially diluted according to a certain concentration and added to a pre-coated elisa plate (CA10 rabbit polyclonal coated 96-well elisa plate), and the binding capacity difference of the antigen evaluation system on the solid virus particles and the hollow virus particles was detected. And under the condition of reaching the same OD value, the reciprocal of the concentration ratio of the added solid virus protein to the added hollow virus protein is the ratio of the reaction capacity of the antigen evaluation system to the solid virus particles to the hollow virus particles. The experimental results show that the antigen evaluation system has 10 times of the capability of reacting with solid virus particles, and the results are shown in Table 2.
TABLE 2 comparison of the reactivity to empty and filled viruses
Example 7 use of antigen evaluation System in vaccine production
In the production stage of the vaccine, the antigen content evaluation system can be adopted to evaluate the antigen content of the samples in the whole process flow. This example focuses on the detection of sucrose density gradient centrifugation tube samples by the system. Through electron microscope observation, 19 # tubes are hollow virus tubes, and 22 # tubes are solid virus tubes. Respectively diluting the antigen standard substance and the sample to certain concentration, adding an enzyme label plate coated with multiple antibodies in advance, performing the steps according to the step of the embodiment 5, making a standard curve by using the OD value of the reference substance and the antigen marking amount, substituting the OD value of the sample into the standard curve to calculate the antigen content of the sample, wherein the detection result is shown in a table 3, and fig. 5 is an evaluation curve graph of the antigen evaluation system for the sample in the super-separation segmentation process.
TABLE 3 evaluation results of antigen evaluation systems applied to ultra-fractionated samples
After the virus liquid is subjected to sucrose density gradient centrifugation, solid virus particles and hollow virus particles are respectively distributed in different sugar degree areas, a 19 # tube is a hollow virus area, and a 22 # tube is a solid virus particle. Table 3 shows the results of ELISA using the antigen evaluation system established in the present invention for antigen detection, and tube 19 shows the peak value of antigen detection, indicating that the system reacts mainly with hollow virus particles.
Example 8 verification of specificity of antigen evaluation System
With reference to the method of example 5, EV71 virus solution, CA16 virus solution, CA10 virus solution, hepatitis a virus solution, poliovirus i-type stock solution, poliovirus ii-type stock solution, poliovirus iii-type stock solution, sample diluent, and CA10 virus purified solution were added, respectively, to verify the specificity of the antigen evaluation system for enterovirus detection.
The result shows that the antigen evaluation system has good specificity and does not react to other types of enteroviruses. The results are shown in Table 4. The results in Table 4 show that the antigen evaluation system has specific specificity for detecting the CA10 virus.
TABLE 4 results of specificity verification of antigen evaluation systems
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Beijing Koxing Bioproduct Co., Ltd
<120> monoclonal antibody of coxsackievirus A10 type hollow virus and application thereof
<130> RYP1910439.9
<160> 16
<170> PatentIn version 3.5
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Claims (16)
1. A monoclonal antibody against CA10 virus, characterized in that it has the heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 5, the heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 6, the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 7, the light chain complementarity determining region CDR1 shown in SEQ ID NO. 13, the light chain complementarity determining region CDR2 shown in SEQ ID NO. 14, and the light chain complementarity determining region CDR3 shown in SEQ ID NO. 15.
2. The monoclonal antibody of claim 1, wherein the monoclonal antibody has a heavy chain having an amino acid sequence set forth in SEQ ID NO. 8; and/or the monoclonal antibody has a light chain with an amino acid sequence shown as SEQ ID NO. 16.
3. The monoclonal antibody of claim 1 or 2, wherein the CA10 virus is CA10 empty virus.
4. The polynucleotide sequence encoding the monoclonal antibody against CA10 virus of any one of claims 1-3, wherein the polynucleotide sequence has the sequence encoding heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 1, the sequence encoding heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 2, the sequence encoding heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 3, and the sequence encoding light chain complementarity determining region CDR1 shown in SEQ ID NO. 9, the sequence encoding light chain complementarity determining region CDR2 shown in SEQ ID NO. 10, the sequence encoding light chain complementarity determining region CDR3 shown in SEQ ID NO. 11.
5. The polynucleotide sequence of claim 4, wherein the polynucleotide sequence has the nucleotide sequence set forth in SEQ ID NO. 4; and/or the polynucleotide sequence has a nucleotide sequence shown in SEQ ID NO. 12.
6. The polynucleotide sequence of claim 4 or 5, wherein the CA10 virus is a CA10 empty virus.
7. A kit for detecting CA10 virus, the kit comprising the monoclonal antibody of any one of claims 1-3;
wherein the CA10 virus is CA10 empty virus.
8. The kit of claim 7, further comprising a polyclonal antibody.
9. The kit of claim 8, wherein said polyclonal antibody is a CA10 rabbit polyclonal antibody.
10. A kit for diagnosing hand-foot-and-mouth disease, characterized in that the kit comprises the monoclonal antibody according to any one of claims 1 to 3.
11. The kit of claim 10, further comprising a polyclonal antibody.
12. The kit of claim 11, wherein said polyclonal antibody is a CA10 rabbit polyclonal antibody.
13. Use of the monoclonal antibody against CA10 virus of any one of claims 1-3 in the preparation of a kit for the detection of CA10 virus or the diagnosis of hand-foot-and-mouth disease;
wherein the CA10 virus is CA10 empty virus.
14. Use of a monoclonal antibody against CA10 virus of any one of claims 1-3 for quality control of the production of a vaccine comprising CA10 virus;
wherein the CA10 virus is CA10 empty virus.
15. A method for quality control of production of a vaccine comprising CA10 virus, the method comprising the step of detecting CA10 virus with a monoclonal antibody against CA10 virus of any one of claims 1 to 3;
wherein the CA10 virus is CA10 empty virus.
16. Use of the monoclonal antibody against the CA10 virus of any one of claims 1-3 for detecting the content of the CA10 virus in the preparation process of a hand-foot-and-mouth vaccine or in a finished vaccine product, wherein the CA10 virus is CA10 empty virus.
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