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Monoclonal Antibody to Coxsackievirus A10 Hollow Virus and Its Application

A monoclonal antibody and virus technology, applied in the field of immunology, can solve the problems of high component requirements, long test time, relatively high equipment requirements, and achieve the effect of enhancing immunogenicity and stability.

Active Publication Date: 2021-08-13
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing process research, the determination of the proportion of hollow and solid viruses in the virus liquid mainly depends on the method of electron microscope observation. The method of electron microscope observation has relatively high requirements on the concentration of the sample and the composition of the buffer system, and the test takes a long time. Equipment requirements are relatively high, with certain limitations

Method used

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  • Monoclonal Antibody to Coxsackievirus A10 Hollow Virus and Its Application
  • Monoclonal Antibody to Coxsackievirus A10 Hollow Virus and Its Application
  • Monoclonal Antibody to Coxsackievirus A10 Hollow Virus and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Preparation of CA10 solid virus particles and hollow virus particles

[0041] Virus cultivation: the CA10 virus strain was used, and the cells used for cultivation were Vero cells (derived from WHO World Health Organization). The cells were cultured in the microcarrier mode of the fermenter tank, and the virus was inoculated into the fermenter tank according to MOI=0.0001~0.001. The culture temperature was 36.0°C±0.5°C. After the virus was cultured for 1-5 days, the CA10 virus liquid was harvested, and the membrane of 100KD-300KD was used. Preliminary purification by clarification and ultrafiltration.

[0042] Sucrose density gradient centrifugation to obtain virus particles with different properties: The method of sucrose density gradient centrifugation was used to separate the hollow and solid of the initially purified CA10 virus liquid and remove impurity proteins. Sucrose gradient of 15% to 60%, centrifuged at 100,000 rpm for 3 to 15 hours. The hollow...

Embodiment 2

[0044] Example 2 Preparation of CA10 Hollow Virus Monoclonal Antibody

[0045] Preparation of hybridoma cell lines: CA10 hollow virus purification solution (preparation method: Vero cells are used to culture CA10 virus, after the virus solution is harvested, it is preliminarily purified and concentrated by clarification and ultrafiltration, and then subjected to sucrose gradient density centrifugation to obtain solid virus. Particle tubes and hollow virus particle tubes, the hollow virus particle tubes are collected and desugared to obtain CA10 hollow virus purified liquid.) Immunize BALB / c mice, 5 for 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: Freund's complete adjuvant for the first injection, incomplete Freund's adjuvant for the second and third injections, and no adjuvant for the fourth injection Antibody; blood test: 1 week after the third dose of immuniza...

Embodiment 3

[0048] Example 3 Purification of CA10 Hollow Virus Monoclonal Antibody

[0049] Antibody purification: centrifuge the ascites prepared from the immunized mice in Example 2 at 2-8°C, 4000-8000 r / min for 5-15 minutes, take the supernatant and filter it through qualitative filter paper, filter it with a 0.45 μm filter membrane, pass through the affinity layer Purified monoclonal antibodies were obtained by analysis. After purification, the detected protein content was 32563 μg / ml. Purified monoclonal antibodies were tested for purity and potency.

[0050] (1) Detection of monoclonal antibody purity

[0051] Perform SDS-PAGE electrophoresis on the purified monoclonal antibody to detect the proportion of IgG heavy chain and light chain, and use a gel imaging scanner to analyze the purity of the monoclonal antibody. See the SDS-PAGE electrophoresis diagram of the purified monoclonal antibody image 3 , tested two groups respectively, in the figure 2 is the diluted sample before p...

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Abstract

The invention discloses a monoclonal antibody capable of reacting with Coxsackie virus A10 hollow virus and a detection kit. The invention adopts purified CA10 virus liquid to immunize mice to prepare monoclonal antibody. The invention also discloses the application of the monoclonal antibody in detecting CA10 virus or diagnosing hand, foot and mouth disease. The monoclonal antibody is widely used in the preparation of rapid detection kits and the development and research of vaccines.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a monoclonal antibody of coxsackie virus A10 hollow virus and application thereof. Background technique [0002] Hand, foot and mouth disease is an acute infectious disease caused by a variety of enterovirus infections. It is prevalent in summer, with a high incidence in preschool children, and adults can be an indirect source of infection. The clinical manifestations of hand, foot and mouth disease are mainly rashes on the mouth, hands, and feet, which can be complicated by meningitis, encephalitis, pulmonary edema, circulatory failure, and other serious illnesses that can lead to death. Currently, enterovirus 71 (EV71) and coxsackievirus A16 (coxsackievirus, CA16) are the most common pathogens causing HFMD in mainland China. However, with the advancement of detection technology and virus typing methods, it has been found that the prevalence of coxsackievirus A6 (coxsackievi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/577G01N33/569A61K39/395A61P31/14
CPCA61P31/14C07K16/1009C07K2317/565C07K2317/76G01N33/56983G01N33/577G01N2333/085G01N2469/10
Inventor 戈小琴武瑞霞安燕秋蔡芳李雅静高强尹卫东
Owner SINOVAC BIOTECH
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