WO2021129246A1 - Monoclonal antibody of coxsackie virus a10 type solid virus and use thereof - Google Patents

Abstract

A monoclonal antibody capable of reacting with a coxsackie virus 10A type solid virus and a detection kit. A purified CA10 virus liquid is used for immunizing mice to prepare the monoclonal antibody. Use of the monoclonal antibody for detecting CA10 virus or diagnosing a hand-foot-and-mouth disease. The monoclonal antibody has wide use in the preparation of a rapid detection kit and development and research of vaccines.

Description

Monoclonal antibody of Coxsackie virus A10 solid virus and its application Technical field

The invention relates to the technical field of immunology, in particular to a monoclonal antibody of Coxsackie virus A10 type solid virus and its application.

Background technique

Hand, foot and mouth disease is an acute infectious disease caused by a variety of enterovirus infections. It is prevalent in summer and has a high incidence in preschool children. Adults can be an indirect source of infection. The main clinical manifestations of hand, foot and mouth disease are rashes on the mouth, hands, and feet, which can be complicated by meningitis, encephalitis, pulmonary edema, circulatory failure and other serious illnesses that lead to death. At present, enterovirus 71 (EV71) and coxsackievirus A16 (coxsackievirus, CA16) are the most common pathogens that cause hand, foot and mouth disease in mainland China. However, with the advancement of detection technology and virus typing methods, in recent years, it has been found that the prevalence of Coxsackievirus A6 (coxsackievirus, CA6) and Coxsackievirus A10 (coxsackievirus, CA10) has been increasing year by year. , And become the main epidemic serotype in some areas. In response to the epidemic trend of viral serotypes in recent years, many domestic and foreign research institutions and companies are focusing on the development of vaccines or drugs that can effectively prevent the occurrence of hand, foot and mouth diseases caused by CA6 or CA10.

In order to provide the fastest plan for clinical treatment, a rapid detection method is usually required. The ELISA rapid detection kit is the most commonly used method, which can detect the virus serotype of the patient in the shortest time. Screening a type-specific monoclonal antibody is a prerequisite for establishing a detection kit. In addition, during the vaccine research process, the antigen content in the vaccine is a key indicator to guide the process research and evaluate the effectiveness in vitro. The most critical technique for establishing an antigen evaluation method is to screen suitable monoclonal antibodies, which can then be used to establish an effective ELISA evaluation system.

Coxsackievirus A6 (coxsackievirus, CA6) and Coxsackievirus A10 (coxsackievirus, CA10) are both enteroviruses. Enteroviruses are prone to form two different structural states when cultured in vitro, one is the integrity of the nucleic acid Solid virus particles, a kind of hollow virus particles that do not contain nucleic acid, usually coexist in these two states. Literature data and many experimental studies have shown that the immunogenicity of solid virus particles is significantly stronger than that of hollow virus particles. In the process of vaccine process research, if the content and ratio of solid virus particles and hollow virus particles in each step of the product can be objectively and truthfully evaluated, it is of great significance to guide the selection of process parameters and the proportioning and measurement of finished products.

Antigen evaluation system (including ELISA rapid detection kit) usually adopts double antibody sandwich method, such as polyclonal antibody (abbreviation: polyclonal antibody)-polyclonal antibody, polyclonal antibody-monoclonal antibody (abbreviation: monoclonal antibody), monoclonal antibody-polyclonal antibody , Monoclonal antibody-the form of monoclonal antibody, the use of polyclonal antibodies usually easily cause cross-binding between different types of enteroviruses, and the specificity of virus detection is not strong; the use of monoclonal antibodies is prone to insufficient epitope binding ability , Can not objectively reflect the true content of antigen.

There is currently no literature report on an antigen evaluation system that can specifically detect CA10 solid virus particles. The current process research to determine the proportion of hollow and solid viruses in the virus liquid mainly relies on the method of electron microscope observation. The method of electron microscope observation requires relatively high sample concentration and the composition of the buffer system, and the test takes a long time. The equipment requirements are relatively high and have certain limitations.

Summary of the invention

In order to solve the above technical problems, the present invention provides the following technical solutions:

In the first aspect, the present invention provides a monoclonal antibody against CA10 virus, the monoclonal antibody having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO: 5, and the heavy chain complementarity determining region shown in SEQ ID NO: 6 Region CDR2, SEQ ID NO: 7 heavy chain complementarity determining region CDR3, and SEQ ID NO: 13 light chain complementarity determining region CDR1, SEQ ID NO: 14 light chain complementarity determining region CDR2, SEQ The light chain complementarity determining region CDR3 shown in ID NO: 15; preferably, the monoclonal antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO: 8; and/or, preferably, the monoclonal antibody has The light chain of the amino acid sequence shown in SEQ ID NO: 16;

Preferably, the CA10 virus is a CA10 solid virus.

In the second aspect, the present invention provides a polynucleotide sequence encoding the monoclonal antibody against CA10 virus, the polynucleotide sequence having the heavy chain complementarity determining region CDR1, SEQ ID NO shown in SEQ ID NO:1 The heavy chain complementarity determining region CDR2 and SEQ ID NO: 3 shown in the heavy chain complementarity determining region CDR3, and the light chain complementarity determining region CDR1 shown in SEQ ID NO: 9 as shown in SEQ ID NO: 10 The light chain complementarity determining region CDR2, the light chain complementarity determining region CDR3 shown in SEQ ID NO: 11; preferably, the polynucleotide sequence has a heavy chain with the nucleotide sequence shown in SEQ ID NO: 4; and /Or, preferably, the polynucleotide sequence has a light chain of the nucleotide sequence shown in SEQ ID NO: 12;

Preferably, the CA10 virus is a CA10 solid virus.

In the third aspect, the present invention provides a kit for detecting CA10 virus, the kit includes the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide sequence; preferably, the kit It also includes polyclonal antibodies; more preferably, the polyclonal antibodies are CA10 rabbit polyclonal antibodies;

Preferably, the CA10 virus is a CA10 solid virus.

In a fourth aspect, the present invention provides a kit for diagnosing hand, foot and mouth disease, the kit comprising the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide sequence; preferably, the reagent The box also includes a polyclonal antibody; more preferably, the polyclonal antibody is a CA10 rabbit polyclonal antibody;

Preferably, the CA10 virus is a CA10 solid virus.

In the fifth aspect, the present invention provides an application of the monoclonal antibody against CA10 virus or the monoclonal antibody encoded by the polynucleotide sequence in the preparation of a kit for detecting CA10 virus or diagnosing hand, foot and mouth disease ；

Preferably, the CA10 virus is a CA10 solid virus.

In a sixth aspect, the present invention provides an application of the monoclonal antibody against the CA10 virus or the monoclonal antibody encoded by the polynucleotide sequence in the quality control of the production of a vaccine containing the CA10 virus;

Preferably, the CA10 virus is a CA10 solid virus.

In the seventh aspect, the present invention provides a method for quality control of the production of a vaccine containing CA10 virus, the method comprising using the monoclonal antibody against the CA10 virus or the monoclonal antibody encoded by the polynucleotide sequence Steps for antibody detection of CA10 virus;

Preferably, the CA10 virus is a CA10 solid virus.

In an eighth aspect, the present invention provides a medicine for treating or preventing diseases caused by CA10 virus infection, the medicine containing the monoclonal antibody against the CA10 virus or the monoclonal antibody encoded by the polynucleotide sequence Antibody;

Preferably, the CA10 virus is a CA10 solid virus.

In the ninth aspect, the present invention provides a monoclonal antibody against CA10 virus or a monoclonal antibody encoded by the polynucleotide sequence for detecting the content of CA10 virus in the preparation of hand, foot and mouth vaccines or in finished vaccines. application.

Preferably, the application is used to detect the content of CA10 solid virus in the preparation process of the hand, foot and mouth vaccine or in the finished vaccine.

The beneficial effects of the present invention are:

The invention provides an antigen evaluation system that mainly detects CA10 solid virus particles. The antigen evaluation system prepared by the monoclonal antibody provided by the present invention can quickly detect solid viruses, and its ability to bind hollow viruses is weak. The proportion of solid virus particles is a key indicator in the vaccine research process and is positively related to the effectiveness of the product. During the virus cultivation stage, a solid system is used to monitor the content of solid virus particles in real time to provide data support for the harvest time of virus cultivation. In the ultracentrifugation purification stage of the virus, the collected solid virus tube can be monitored through the solid system, which provides guidance for the subsequent ultracentrifugation and merger. The use of the antigen evaluation system of the present invention can not only provide guidance for the vaccine process research, but also quickly evaluate the effectiveness of the product.

Of course, the antigen evaluation system can also be applied to the preparation of rapid detection kits and the research and development of vaccines.

Description of the drawings

Figure 1 is a photograph of the centrifuge tube after sucrose density gradient centrifugation in Example 1;

Figure 2 is an electron microscope comparison diagram of solid virus particles and hollow virus particles identified by electron microscopy in Example 1. In the figure: the left picture is tube 22 solid virus particles, and the right picture is tube 19 hollow virus particles;

Figure 3 is an SDS-PAGE electrophoresis diagram of the monoclonal antibody in Example 3 after purification. In the figure: 2-before purification, 3-purification eluent (heavy chain + light chain), 4-purification flow-through;

Figure 4 is a linear relationship diagram of the antigen evaluation system of Example 5;

FIG. 5 is an evaluation curve diagram of samples used in the ultra-isolation segmentation process by the antigen evaluation system of Example 7. FIG.

Sequence Listing Description

Note: The underlined font part is the CDR area, and the bold font part is the CH1 end primer.

Detailed ways

The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present 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 CA10 hollow virus particles and solid virus particles

Virus culture: CA10 virus strain is used, and the culture cells are Vero cells (from WHO World Health Organization). Cells are cultured in a fermenter microcarrier mode, the virus is inoculated into the fermentor at MOI = 0.0001～0.001, the culture temperature is 36.0℃±0.5℃, the virus is cultured for 1 to 5 days and then harvested to obtain CA10 virus liquid, using 100KD～300KD membrane Package clarification and ultrafiltration preliminary purification.

Sucrose density gradient centrifugation to obtain virus particles of different properties: The sucrose density gradient centrifugation method is used to carry out hollow separation and remove impurity proteins from the preliminary purified CA10 virus liquid. The sucrose gradient is 15%-60%, and centrifuged at 100,000 rpm for 3-15 hours. The solid virus band and the hollow virus band were separated by sucrose density gradient centrifugation, and the two virus bands were taken out respectively, and the results of the hollow and hollow separation were identified by an electron microscope.

The photograph of the centrifuge tube after sucrose density gradient centrifugation is shown in Figure 1. From the figure, the separated solid virus bands and hollow virus bands can be seen. The electron microscope comparison diagram of hollow virus particles and solid virus particles identified by electron microscope is shown in Figure 2.

Example 2 Preparation of CA10 solid virus monoclonal antibody

Hybridoma cell line preparation: use CA10 solid virus purification solution (preparation method of solid virus purification solution: Vero cells culture CA10 virus, after harvesting the virus solution, clarifying and ultrafiltration, preliminary purification and concentration, sucrose gradient density centrifugation, solid virus can be obtained Particle tube and hollow virus particle tube, the solid virus particle tube is collected and then desugared to obtain the CA10 solid virus purified solution.) Immune BALB/c mice, immunize 5 mice of each type; at 0, 2, 4, and 6 weeks A total of 4 injections of subcutaneous multi-point immunization on the back; immunization dose: 0.2ml/needle/head; adjuvant: the first injection is Freund's complete adjuvant, the second and third injections are Freund's incomplete adjuvant, and the fourth injection is not adjuvanted agent; blood test: after the third immunization blood test pin 1 week indirect ELISA titers of antibody titer of more than 10 ⁴ 5 mice were boosted by intraperitoneal injection needle.

Cell fusion: 3 days after the booster immunization was injected into the abdominal cavity, the mice were sacrificed and the spleens were fused, and after two monoclonalizations to obtain a positive hybridoma cell line (OD450 value of cell supernatant>1.0), the mice were immunized to prepare ascites.

The cryopreserved CA10 solid virus murine monoclonal hybridoma cell line was removed from liquid nitrogen for resuscitation and expansion. The total nucleic acid was extracted when the amount was more than ^{10 6 and was entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. to perform PCR amplification of the monoclonal antibody.} The chain and light chain sequences are sequenced, and then the corresponding amino acid sequence is determined by the nucleotide sequence. For the determined polynucleotide sequence and amino acid sequence, please refer to the sequence table.

Example 3 Purification of CA10 solid virus monoclonal antibody

Antibody purification: Centrifuge the ascites prepared by immunized mice in Example 2 at 2～8℃, 4000～8000r/min for 5～15 minutes, take the supernatant and filter it with qualitative filter paper, filter it with 0.45μm filter membrane, and pass it through the affinity layer Analyze to obtain purified monoclonal antibody. After purification, the protein content was 3334μg/ml. Purified monoclonal antibodies were tested for purity and titer.

(1) Detection of monoclonal antibody purity

The purified monoclonal antibody was subjected to SDS-PAGE electrophoresis to detect the proportion of IgG heavy chain and light chain. The purity of the monoclonal antibody was analyzed by a gel imaging scanner. The SDS-PAGE electrophoresis diagram of the purified monoclonal antibody is shown in Figure 3.

(2) Determination of monoclonal antibody titer

Pre-coating: CA10 purified solution is diluted to 0.5-2.0μg/ml with 0.01M phosphate buffer, and coated with 96-well microtiter plate at 4°C overnight or 37°C for 2 hours. Add a 0.01M phosphate buffer solution with a final concentration of 0.05% Tween 20 to wash 2 to 5 times, then add a 0.01M phosphate buffer solution containing 5-20% calf serum and block at 37°C for 1 to 2 hours. Remove the blocking solution and beat the plate to remove the remaining blocking solution in the well.

Potency: the tested serum and negative control sera are serially diluted ^108-fold gradient of 10 times according to the gradient method, were added to 10 ² 10 ⁸ dilution of the sample to the 96-well plate, each well was added 100μl, 37 ℃ incubated For 0.5 to 2 hours, wash 2 to 5 times with 0.01M PBST20 washing solution, add anti-mouse IgG HRP (commercially available: KPL manufacturer, directly diluted 12000 times after purchase), incubate at 37°C for 0.5-2 hours, wash plate 2 -After 5 times, pat dry, add color-developing substrate and develop color at 37°C for 8-15 minutes, _{stop with 2M H 2} SO ₄ , and read at 450nm wavelength. Standard: Under the same dilution factor, the sample OD450 value ≥ negative control × 2.1, if the negative control absorbance value is less than 0.05, calculate with 0.05 to determine the positive standard. The titer detected by indirect ELISA method was 10 ⁷ .

In Figure 3, the ratio of the Marker protein band to the total protein in the lane is shown in the table below.

To	1	2	3	4	5	6	7	8	9
Mark	0.1339	0.1102	0.1239	0.1945	0.0882	0.2443	0.0363	0.0722	0.0040

The proportions of the heavy chain and the light chain of the purified eluate obtained by the detection are respectively: the heavy chain accounts for 0.6880 and the light chain accounts for 0.3120. From this result, the sum of the ratio of heavy chain to light chain in the purified monoclonal antibody eluate is 100%, indicating that the purification effect is very good, and there is no impurity protein.

Example 4 Labeling enzyme of CA10 solid virus monoclonal antibody

Place the purified monoclonal antibody in a dialysis bag, dialyze it in a 0.05M carbonate buffer system for 2-8 hours, and change the dialysate every 1-2 hours; use sodium periodate to activate HRP, 20% ethyl acetate Diol terminates activation. The activated HRP was added to the antibody, and the dialysis was continued overnight; the antibody was taken out to obtain the HRP-labeled CA10 solid virus monoclonal antibody, which was reduced by adding sodium borohydride; after precipitation with ammonium sulfate, it was reconstituted with 0.01MPBS and stored below -20°C.

Example 5 Antigen Evaluation System Matching

CA10 rabbit polyclonal antibody (manufactured by Beijing Kexing Biological Products Co., Ltd., referred to as Beijing Kexing) was diluted with a carbonate buffer solution according to a certain ratio, and then coated with a 96-well microtiter plate at 4°C overnight or 37°C for 2 hours. Add a 0.01M phosphate buffer solution with a final concentration of 0.05% Tween 20 to wash 2 to 5 times, then add a 0.01M phosphate buffer solution containing 5-20% calf serum and block at 37°C for 1 to 2 hours. Remove the blocking solution and beat the plate to remove the remaining blocking solution in the well. The technical solution involved in the present invention can be made into an enzyme-labeled dry plate in advance when the kit is made, and incubated at 2-8°C.

Antigen dilution: The CA10 virus solution is serially diluted according to a certain concentration, 80U/ml, 40U/ml, 20U/ml, 10U/ml, 5U/ml are sequentially added to the above 96-well plate, 100μl per well, and incubated at 37°C for 0.5 After -2 hours, wash 2 to 5 times with 0.01M PBST20 washing solution, add the HRP-labeled CA10 solid virus monoclonal antibody prepared in Example 4, incubate at 37°C for 0.5-2 hours, wash the plate 2-5 times, Pat dry, add the chromogenic substrate and develop color at 37°C for 8-15 minutes, _{stop with 2M H 2} SO ₄ , and read at the wavelength of 450-630 nm. Investigate the sensitivity and linear relationship of the antigen evaluation system.

The detection sensitivity of the antigen evaluation system to CA10 virus is 5U/ml, and the linear correlation R ² ≥0.98. The results are shown in Table 1, and the linear relationship diagram of the antigen evaluation system is shown in Figure 4.

Table 1 Sensitivity test results

Viral antigen (U/ml)	OD value 1	OD value 2	Mean OD
Blank hole	0.069	0.073	0.071
5	0.175	0.172	0.174
10	0.301	0.277	0.289
20	0.562	0.525	0.543
40	1.042	0.967	1.005
80	1.944	1.838	1.891

Example 6 Evaluation of the binding ability of the antigen evaluation system to hollow virus particles or solid virus particles

Referring to the method in Example 5, CA10 hollow virus particles (from Beijing Kexing) and CA10 solid virus particles (from Beijing Kexing) were serially diluted at a certain concentration and then added to the pre-coated ELISA plate (CA10 rabbit Multi-antibody-coated 96-well ELISA plate) to detect the difference in the binding ability of the above-mentioned antigen evaluation system to hollow virus particles and solid virus particles. Under the condition that the same OD value is reached, the reciprocal of the ratio of the added hollow virus and solid virus protein concentration is the ratio of the response ability of the antigen evaluation system to hollow virus particles and solid virus particles. The experimental results show that the reaction ability of the antigen evaluation system to solid virus particles is 80 times that of hollow virus particles. The results are shown in Table 2.

Table 2 Comparison results of response ability to solid virus and hollow virus

Example 7 Application of Antigen Evaluation System in Vaccine Production

In the vaccine production stage, the antigen evaluation system can be used to evaluate the antigen content of samples in the entire process. This example focuses on the system's detection of samples in each tube of sucrose density gradient centrifugation. Observed by electron microscope, tube 19 is a solid virus tube, and tube 22 is a hollow virus tube. Dilute the antigen standard and the sample to a certain concentration respectively, add to the pre-coated ELISA plate with many antibodies, follow the steps in Example 5, make a standard curve from the OD value of the reference product and the labeled amount of the antigen, and the sample OD value band Enter the standard curve to calculate the antigen content of the sample. The test results are shown in Table 3. Figure 5 shows the evaluation curve of the sample used by the antigen evaluation system in the ultra-isolation segmentation process.

Table 3 Evaluation results of the antigen evaluation system applied to ultra-isolated segmented samples

After the virus solution is centrifuged with a sucrose density gradient, the solid virus particles and hollow virus particles are respectively distributed in areas of different sugar content, tube 19 is the hollow virus region, and tube 22 is the solid virus particle. Table 3 shows the ELISA results of antigen detection using the antigen system established by the present invention. Tube 22 is the peak value of antigen detection, indicating that the system mainly reacts with solid virus particles.

Example 8 Verification of the specificity of the antigen evaluation system

According to the method of Example 5, add EV71 virus solution, CA16 virus solution, CA10 virus solution, hepatitis A virus solution, poliovirus type I stock solution, poliovirus type Ⅱ stock solution, poliovirus type Ⅲ stock solution, and sample dilution. CA10 virus purification solution, CA10 infected mouse feces samples (feces samples are diluted with PBS buffer and centrifuged to collect the supernatant) and CA10 infected mouse serum samples (eyeball blood sampling, centrifugal separation of serum), verify that the antigen evaluation system is effective in intestinal The specificity of road virus detection.

The results show that the antigen evaluation system has good specificity and does not respond to other types of enteroviruses. The results are shown in Table 4. The results in Table 4 show that this antigen evaluation system has exclusive specificity for CA10 virus detection.

Table 4 Specificity verification results of the antigen evaluation system

Although the general description, specific embodiments and experiments have been used to describe the present invention in detail above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.

Claims (10)

1. A monoclonal antibody against the CA10 virus, characterized in that the monoclonal antibody has the heavy chain complementarity determining region CDR1 shown in SEQ ID NO: 5, and the heavy chain complementarity determining region CDR2 shown in SEQ ID NO: 6; The heavy chain complementarity determining region CDR3 shown in NO: 7 and the light chain complementarity determining region CDR1 and SEQ ID NO: 14 shown in SEQ ID NO: 13 and the light chain complementarity determining region CDR2 and SEQ ID NO: 15. The light chain complementarity determining region CDR3 shown in FIG.; preferably, the monoclonal antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO: 8; and/or, preferably, the monoclonal antibody has SEQ ID NO: 16 The light chain of the amino acid sequence shown;

   Preferably, the CA10 virus is a CA10 solid virus.
2. The polynucleotide sequence encoding the monoclonal antibody against CA10 virus of claim 1, wherein the polynucleotide sequence has the heavy chain complementarity determining regions CDR1 and SEQ ID NO shown in SEQ ID NO: 1. The heavy chain complementarity determining region CDR2, SEQ ID NO: 3 shown in the heavy chain complementarity determining region CDR3, and the light chain complementarity determining region CDR1 shown in SEQ ID NO: 9 shown in SEQ ID NO: 10 Light chain complementarity determining region CDR2, light chain complementarity determining region CDR3 shown in SEQ ID NO: 11; preferably, the polynucleotide sequence has a heavy chain with the nucleotide sequence shown in SEQ ID NO: 4; and/ Or, preferably, the polynucleotide sequence has a light chain of the nucleotide sequence shown in SEQ ID NO: 12;

   Preferably, the CA10 virus is a CA10 solid virus.
3. A kit for detecting CA10 virus, wherein the kit comprises the monoclonal antibody according to claim 1 or the monoclonal antibody encoded by the polynucleotide sequence according to claim 2; preferably, the reagent The box also includes a polyclonal antibody; more preferably, the polyclonal antibody is a CA10 rabbit polyclonal antibody;

   Preferably, the CA10 virus is a CA10 solid virus.
4. A kit for diagnosing hand, foot and mouth disease, wherein the kit comprises the monoclonal antibody of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2; preferably, the The kit also includes a polyclonal antibody; more preferably, the polyclonal antibody is a CA10 rabbit polyclonal antibody;

   Preferably, the CA10 virus is a CA10 solid virus.
5. Use of the monoclonal antibody against CA10 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2 in the preparation of a kit for detecting CA10 virus or diagnosing hand, foot and mouth disease;

   Preferably, the CA10 virus is a CA10 solid virus.
6. The use of the monoclonal antibody against CA10 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2 in the quality control of the production of vaccines containing CA10 virus;

   Preferably, the CA10 virus is a CA10 solid virus.
7. A method for quality control of the production of a vaccine containing CA10 virus, characterized in that the method comprises using the monoclonal antibody against the CA10 virus according to claim 1 or the monoclonal antibody encoded by the polynucleotide sequence according to claim 2 Steps of cloning antibody to detect CA10 virus;

   Preferably, the CA10 virus is a CA10 solid virus.
8. A medicine for the treatment or prevention of diseases caused by CA10 virus infection, characterized in that the medicine contains the monoclonal antibody against CA10 virus according to claim 1 or the single nucleotide sequence encoded by the polynucleotide sequence according to claim 2 Cloned antibody

   Preferably, the CA10 virus is a CA10 solid virus.
9. The monoclonal antibody against CA10 virus according to claim 1 or the monoclonal antibody encoded by the polynucleotide sequence according to claim 2 is used for detecting the content of CA10 virus in the preparation process of hand, foot and mouth vaccines or in finished vaccines.
10. The application according to claim 9, characterized in that the application is used to detect the content of CA10 solid virus in the preparation process of the hand, foot and mouth vaccine or in the finished product of the vaccine.

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