CN110938141A

CN110938141A - Monoclonal antibody of coxsackievirus A6 type hollow virus and application thereof

Info

Publication number: CN110938141A
Application number: CN201911383201.3A
Authority: CN
Inventors: 武瑞霞; 戈小琴; 刘亚茹; 蔡芳; 李雅静; 朱朗; 高强; 尹卫东
Original assignee: Sinovac Biotech Co ltd
Current assignee: Sinovac Biotech Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-03-31
Anticipated expiration: 2039-12-27
Also published as: CN110938141B

Abstract

The invention discloses a monoclonal antibody capable of reacting with coxsackievirus A6 type hollow virus, and discloses a kit containing the monoclonal antibody and application thereof. The invention adopts purified CA6 virus liquid to immunize mice to prepare monoclonal antibodies. The invention also discloses application of the monoclonal antibody in detecting CA6 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

Monoclonal antibody of coxsackievirus A6 type hollow virus and application thereof

Technical Field

The invention relates to the technical field of immunology, in particular to a monoclonal antibody of a coxsackievirus A6 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.

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 CA6 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 CA6 virus is a CA6 type empty virus.

In a second aspect, the present invention provides a polynucleotide sequence encoding the monoclonal antibody against CA6 virus, the polynucleotide sequence having a heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 1, a heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 2, a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 3, and a light chain complementarity determining region CDR1 shown in SEQ ID NO. 9, a light chain complementarity determining region CDR2 shown in SEQ ID NO. 10, a light chain complementarity determining region CDR3 shown in SEQ ID NO. 11; preferably, the polynucleotide sequence has the heavy chain of the nucleotide sequence shown in SEQ ID NO. 4; and/or, preferably, the polynucleotide sequence has the nucleotide sequence shown in SEQ ID NO. 12 as the light chain;

preferably, the CA6 virus is a CA6 type empty virus.

In a third aspect, the invention provides a kit for detecting the CA6 virus, the kit comprising the monoclonal antibody or a monoclonal antibody encoded by the polynucleotide sequence; preferably, the kit further comprises a polyclonal antibody; more preferably, the polyclonal antibody is a CA6 rabbit polyclonal antibody;

preferably, the CA6 virus is a CA6 type 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 CA6 rabbit polyclonal antibody;

preferably, the CA6 virus is a CA6 type empty virus.

In a fifth aspect, the invention provides an application of the monoclonal antibody against the CA6 virus or the monoclonal antibody coded by the polynucleotide sequence in preparing a kit for detecting the CA6 virus or diagnosing hand-foot-and-mouth disease;

preferably, the CA6 virus is a CA6 type empty virus.

In a sixth aspect, the invention provides the use of the monoclonal antibody against CA6 virus or the monoclonal antibody encoded by the polynucleotide sequence for quality control of the production of a vaccine comprising CA6 virus;

preferably, the CA6 virus is a CA6 type empty virus.

In a seventh aspect, the invention provides a method of quality control of production of a vaccine comprising CA6 virus, said method comprising the step of detecting CA6 virus with said monoclonal antibody against CA6 virus or said monoclonal antibody encoded by a polynucleotide sequence;

preferably, the CA6 virus is a CA6 type empty virus.

In an eighth aspect, the invention provides a medicament for treating or preventing a disease caused by infection with CA6 virus, said medicament comprising said monoclonal antibody against CA6 virus or said monoclonal antibody encoded by said polynucleotide sequence;

preferably, the CA6 virus is a CA6 type empty virus.

In a ninth aspect, the invention provides an application of the monoclonal antibody aiming at the CA6 virus or the monoclonal antibody coded by the polynucleotide sequence in detecting the content of the CA6 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 the CA6 hollow virus in the preparation process of the hand-foot-and-mouth vaccine or in the vaccine finished product.

At present, no antigen evaluation system capable of mainly detecting CA6 hollow virus particles is reported in the literature. 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.

The invention adopts purified CA6 virus liquid to immunize a mouse to prepare a monoclonal antibody, immunizes a rabbit to prepare a polyclonal antibody, screens out a cell strain with strong reaction capability with CA6 type hollow virus particles from a plurality of monoclonal cell strains, and utilizes the cell strain to prepare a large amount of ascites. Then, the monoclonal antibody and the polyclonal antibody are respectively purified, and then the purified monoclonal antibody is labeled with HRP enzyme, so that a set of antigen detection system which has stronger reaction capability with hollow virus particles than solid virus particles is established with the prepared polyclonal antibody, namely an antigen evaluation system which is mainly used for detecting CA6 hollow virus particles.

Preparing polyclonal antibody and monoclonal antibody with type specific binding capacity with CA6 type hollow virus particles, establishing an antigen evaluation system capable of specifically detecting CA6 virus, wherein the binding capacity of the evaluation system to CA6 type hollow virus particles is obviously stronger than that of CA6 type solid virus particles. By adopting the antigen evaluation system, the content of the CA6 type hollow virus particles in the sample can be rapidly analyzed within 4 hours after the sample is taken, and the packaging conditions of the CA6 type hollow virus particles and the CA6 type solid virus particles in the virus liquid can be evaluated at different time points of virus culture by combining a single CA6 type solid virus detection system, so that the antigen evaluation system has important significance for guiding the virus harvesting time point in process research. In the virus ultracentrifugation process, a hollow antigen detection system and a solid antigen detection system are adopted for evaluation respectively, so that a main CA6 type hollow virus tube and a main CA6 type solid virus tube can be distinguished quickly, and the virus ultracentrifugation process can provide data support for virus ultraclutching.

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 detection 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 development and research of vaccines. Antigen evaluation systems (including ELISA rapid detection kits) usually employ double antibody sandwich methods, such as polyclonal antibody (polyclonal antibody), polyclonal antibody, monoclonal antibody (monoclonal antibody), monoclonal antibody-polyclonal antibody, monoclonal antibody-monoclonal antibody.

Drawings

FIG. 1 is a photograph of a centrifuge tube after sucrose density gradient centrifugation in example 1;

FIG. 2 is an electron microscope comparison image of solid viral particles and hollow viral particles identified by the electron microscope of example 1, wherein the left image is an electron microscope image of a No. 23 tube solid viral particle, and the right image is an electron microscope image of a No. 19 tube hollow viral 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 of the evaluation of the antigen evaluation system of example 7 for a sample from the superseparation segmentation process.

Description of sequence listing

Note: the underlined part is the CDR region and the bold 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 CA6

And (3) culturing the virus: the CA6 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 CA6 virus liquid, and clarifying and ultrafiltering for primary purification by adopting a membrane package with the density of 100 KD-300 KD.

Sucrose density gradient centrifugation yields viral particles of different nature: and (3) performing air-solid separation and impurity protein removal on the CA6 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 CA6 hollow Virus

Preparing hybridoma cell strains: a CA6 hollow virus purified solution is adopted (the preparation method of the hollow virus purified solution is that Vero cells culture CA6 virus, after the virus solution is harvested, 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 desugarized to obtain the CA6 hollow virus purified solution) to immunize BALB/c mice, wherein 5 mice are immunized per type; 5 needles in total for subcutaneous dorsal multipoint immunization at 0, 2, 4, 6, 8 weeks; immunization dose: 0.2 ml/needle/stick; adjuvant: the 1 st needle is Freund's complete adjuvant, the 2 nd, 3 th and 4 th needles are Freund's incomplete adjuvant, and the 5 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 boosted by intraperitoneal injection through the 5 th needle.

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 CA6 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 CA6

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. The protein content after purification was 3334. 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).

The proportion of Marker (Marker) protein bands in FIG. 3 to total protein in the lanes is shown in Table 1 below.

TABLE 1

The proportions of the heavy chain and the light chain of the purified eluent obtained by detection are 0.6127 for the heavy chain and 0.3873 for the light chain respectively. 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.

(2) Determination of the potency of the monoclonal antibody

Pre-coating: and diluting the CA6 purified solution (obtained by inoculating Vero cells with CA6 virus, obtaining the virus, clarifying and ultrafiltering, centrifuging with sucrose gradient density and desugaring) to 0.5-2.0 mu g/ml by using 0.01M phosphate buffer solution, and coating a 96-hole enzyme label plate 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: serial gradient dilution of the serum to be detected and the negative serum contrast is carried out to 10 times according to a 10-fold gradient method⁸Adding 10 times of²To 10⁸Adding 100 μ l of diluted sample into the 96-well plate, incubating at 37 deg.C for 0.5-2 hr, washing with 0.01MPBST20 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 developing 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。

Example 4 Standard enzyme of the hollow Virus monoclonal antibody CA6

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 CA6 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 CA6 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 CA6 virus liquid according to a certain concentration, sequentially adding 80U/ml, 40U/ml, 20U/ml, 10U/ml and 5U/ml into the 96-well plate, adding 100 mu l of the mixture into each well, incubating for 0.5-2 hours at 37 ℃, washing for 2-5 times by 0.01MPBST20 washing liquid, adding the HRP-labeled CA6 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 2-2M H minutes₂SO₄The reading is terminated at the wavelength of 450-630 nm. And (3) inspecting the sensitivity and linear relation of the antigen system.

The detection sensitivity of the antigen detection system to the CA6 virus is 5U/ml, and the linear correlation R²The result is more than or equal to 0.98, the result is shown in Table 2, and the linear relation graph of the antigen detection system is shown in FIG. 4.

TABLE 2 sensitivity test results

Virus antigen (U/ml)	OD value 1	OD value 2	OD mean value
				Blank hole	0.054	0.062	0.058
5	0.143	0.140	0.142
				10	0.267	0.271	0.269
20	0.514	0.531	0.523
				40	0.984	0.982	0.983
80	2.054	2.101	2.078

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, CA6 solid virus particles (from beijing kakking) and CA6 hollow virus particles (from beijing kakking) were serially diluted according to a certain concentration and added to a pre-coated elisa plate (CA6 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 8 times of the capability of reacting with solid virus particles, and the results are shown in Table 3.

TABLE 3 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 4, and fig. 5 is an evaluation curve graph of an antigen system for a sample in a super-separation segmentation process.

TABLE 4 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 regions, wherein 19 # tubes are hollow virus particles, and 22 # tubes are solid virus particles. Table 4 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

Referring 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 CA6 virus purified solution were added, respectively, to verify the specificity of the antigen detection system in 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 5. The results in Table 5 show that the antigen evaluation system has specific specificity for detecting CA6 virus.

TABLE 5 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 A6 type hollow virus and application thereof

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Claims

1. A monoclonal antibody against CA6 virus, characterized in that it has a heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 5, a heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 6, a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 7, a light chain complementarity determining region CDR1 shown in SEQ ID NO. 13, a light chain complementarity determining region CDR2 shown in SEQ ID NO. 14, and a 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 CA6 virus is a CA6 type empty virus.

2. A polynucleotide sequence encoding the monoclonal antibody against CA6 virus of claim 1, wherein the polynucleotide sequence has a heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 1, a heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 2, a heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 3, and a light chain complementarity determining region CDR1 shown in SEQ ID NO. 9, a light chain complementarity determining region CDR2 shown in SEQ ID NO. 10, a light chain complementarity determining region CDR3 shown in SEQ ID NO. 11; preferably, the polynucleotide sequence has the heavy chain of the nucleotide sequence shown in SEQ ID NO. 4; and/or, preferably, the polynucleotide sequence has the nucleotide sequence shown in SEQ ID NO. 12 as the light chain;

preferably, the CA6 virus is a CA6 type empty virus.

3. A kit for detecting CA6 virus, the kit comprising the monoclonal antibody of claim 1 or a monoclonal antibody encoded by the polynucleotide sequence of claim 2; preferably, the kit further comprises a polyclonal antibody; more preferably, the polyclonal antibody is a CA6 rabbit polyclonal antibody;

preferably, the CA6 virus is a CA6 type empty virus.

4. A kit for diagnosing hand-foot-and-mouth disease, comprising the monoclonal antibody of claim 1 or a monoclonal antibody encoded by the polynucleotide sequence of claim 2; preferably, the kit further comprises a polyclonal antibody; more preferably, the polyclonal antibody is a CA6 rabbit polyclonal antibody;

preferably, the CA6 virus is a CA6 type empty virus.

5. Use of the monoclonal antibody against CA6 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2 in the preparation of a kit for the detection of CA6 virus or the diagnosis of hand-foot-and-mouth disease;

preferably, the CA6 virus is a CA6 type empty virus.

6. Use of a monoclonal antibody against the CA6 virus of claim 1 or a monoclonal antibody encoded by the polynucleotide sequence of claim 2 for quality control of the production of a vaccine comprising the CA6 virus;

preferably, the CA6 virus is a CA6 type empty virus.

7. A method for quality control of production of a vaccine comprising CA6 virus, the method comprising the step of detecting CA6 virus with the monoclonal antibody against CA6 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2;

preferably, the CA6 virus is a CA6 type empty virus.

8. A medicament for treating or preventing a disease caused by infection with CA6 virus, said medicament comprising the monoclonal antibody against CA6 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2;

preferably, the CA6 virus is a CA6 type empty virus.

9. Use of the monoclonal antibody against CA6 virus of claim 1 or the monoclonal antibody encoded by the polynucleotide sequence of claim 2 for detecting the content of CA6 virus in the process of preparing a hand-foot-and-mouth vaccine or in the finished vaccine product.

10. The use according to claim 9, wherein the use is for detecting the content of CA6 empty virus in the preparation process of a hand-foot-and-mouth vaccine or in a vaccine finished product.