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Coxsackie virus group A 16-type monoclonal antibody 16E1 and preparation method thereof

A monoclonal antibody and Coxsackie virus technology, applied in the field of antibody drugs and virology, can solve the problem of lack of CA16 antigen detection kits, etc., and achieve excellent application significance and good specificity

Inactive Publication Date: 2020-01-14
BEIJING WANTAI BIOLOGICAL PHARMACY ENTERPRISE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The detection of viral antigen content is an important means of quality control in the vaccine production process, but there is still a lack of a unified CA16 antigen detection kit

Method used

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  • Coxsackie virus group A 16-type monoclonal antibody 16E1 and preparation method thereof
  • Coxsackie virus group A 16-type monoclonal antibody 16E1 and preparation method thereof
  • Coxsackie virus group A 16-type monoclonal antibody 16E1 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of anti-CA16 monoclonal antibody

[0049] (1) Immunization of mice

[0050] Immunization of CA16 virus: BALB / c female mice aged 6-8 weeks were immunized with CA16 virus immunogen, and boosted immunization every 2 weeks. Before each immunization, 200 μL of eye socket vein blood or 20 μL of tail vein blood were collected for preparation Titer determination. When the serum titer of the mice reached the plateau, the immunization was stopped, and fusion was carried out after two months of rest.

[0051] (2) Preparation and screening of fusion hybridomas

[0052] 1. Preparation of mouse macrophages: A 6-week-old BALB / c mouse was killed, rinsed with tap water, and soaked in 75% ethanol solution for 5 minutes; the mouse was taken out and the abdomen was fully exposed, and sterile Lift the peritoneum with tweezers, cut a small opening in the center of the peritoneum, inject an appropriate amount of HT culture solution into the abdominal cavity through t...

Embodiment 3

[0071] Example 3: Preparation of CA16 HRP-labeled rabbit polyclonal antibody

[0072] The Vero cells were recovered and cultured at 36.5±0.5°C. The cell concentration reaches 0.1~10×10 6 Infection of Vero cells with CA16 virus (CA16V-24) at an MOI of 0.05 to 0.1, cultured at 35.5±0.5°C for 2 to 4 days, and the cell supernatant was harvested, which was the CA16 harvest solution. The CA16 harvest liquid was clarified and then concentrated more than 5 times by ultrafiltration membrane. Then carry out molecular sieve chromatography and ion exchange chromatography, the detection wavelength is 280nm, collect eluent and flow-through respectively to obtain purified solution, and obtain CA16 vaccine stock solution after formaldehyde inactivation. The purity of the CA16 purified liquid is over 90% as detected by HPLC. The detected antigen content was 13220U / ml, and the total protein content was 6μg / ml. Use this stock solution as the immunogen.

[0073] The immunized animals were 2...

Embodiment 4

[0083] Embodiment 4: Determination of antigen detection system

[0084] Three commonly used ELISA detection systems were compared, namely:

[0085] System 1: rabbit polyclonal antibody + antigen + 16E1 (mouse) + goat anti-rabbit HRP

[0086] System 2: 16E1 (mouse) + antigen + rabbit polyclonal antibody + goat anti-rabbit HRP

[0087] System 3: 16E1 (mouse) + antigen + rabbit polyclonal antibody HRP.

[0088] Adopt the checkerboard method to explore the appropriate concentration for use, and the antigen is the CA16 vaccine stock solution produced according to Example 1.

[0089] In the case of meeting the background requirements, the sensitivity of system 1 can only reach 125ng / ml, which cannot meet the detection requirements; the sensitivity of system 2 is the highest, reaching 1ng / ml; the sensitivity of system 3 can reach 12.5ng / ml. Since system 3 has fewer detection steps and can shorten the detection time, system 3 was chosen to establish the detection method.

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Abstract

The invention provides a coxsackie virus group A 16-type monoclonal antibody 16E1. The monoclonal antibody is generated by a hybridoma cell strain 16E1, the hybridoma cell strain is preserved in ChinaCenter for Type Culture Collection on August 1, 2019, and a preservation number of the hybridoma cell strain is CCTCC No. C2019167. The 16E1 monoclonal antibody provided by the invention has good specificity, does not generate cross reaction with other types of enteroviruses, has good broad spectrum for CA16 viruses from different sources, and has excellent application significance.

Description

【Technical field】 [0001] The present invention relates to the technical fields of virology and antibody medicine, in particular to a Coxsackievirus A group 16 monoclonal antibody 16E1, a hybridoma cell line producing the monoclonal antibody, and a construction method and application of the cell line. 【Background technique】 [0002] Hand, foot and mouth disease is one of the Class C infectious diseases in my country. The initial manifestations are fever, rashes, herpes, and small ulcers on the hands, feet, and mouth. Some patients will develop severe symptoms such as aseptic encephalitis and meningitis. brought about a large burden of disease. At present, there are reports of HFMD cases in most parts of the world, and the Asia-Pacific region, as an area with a high incidence of HFMD, has received sufficient attention in recent years. Enterovirus 71 (EV71) and Coxsackievirus A group 16 (CA16) are the main pathogens causing HFMD, although in recent years Coxsackievirus A group ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20G01N33/569
CPCC07K16/1009G01N33/56983G01N2333/085
Inventor 程通叶祥忠毛群颖徐龙发梁争论高帆韩金乐夏宁邵
Owner BEIJING WANTAI BIOLOGICAL PHARMACY ENTERPRISE
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