Anti-Japanese encephalitis virus monoclonal antibody and its application

A technology of monoclonal antibody and Japanese encephalitis virus, applied in the biological field, can solve the problems of long time for virus isolation and culture, unsatisfactory results of serological diagnosis of Japanese encephalitis, and low positive rate

Inactive Publication Date: 2011-12-21
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Due to the extensive serological cross-reaction among flaviviruses, some patients have not yet produced antibodies in the early stage of the disease, so that the serological diagnosis of JE cannot achieve satisfactory results; the toxemia period of JE patients is very short, and the time for virus isolation and culture is long, and the positive rate is low. The generation of low RT-PCR technology provides an early, rapid, sensitive and specific method for virus detection

Method used

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  • Anti-Japanese encephalitis virus monoclonal antibody and its application
  • Anti-Japanese encephalitis virus monoclonal antibody and its application
  • Anti-Japanese encephalitis virus monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 prepares pure whole virus antigen

[0040] Step 1 Virus Propagation

[0041] Use BHK cells (Shanghai Jinke Biological Co., Ltd.) to grow into cells in DMEM containing 8% calf serum (Hangzhou Sijiqing Bioengineering Materials Co., Ltd.), 100 U / ml penicillin, and 100 μg / ml streptomycin Pour off the nutrient solution after the single layer, wash once with PBS (pH7.2) or serum-free nutrient solution, inoculate with JEV (SA14-14-2, Hunan Zhongan Biopharmaceutical Co., Ltd.), and treat at 37°C for 1.5-2h , then washed once with PBS (NaCl 137mmol / L, KCl 2.7mmol / L, Na2HPO4 4.3mmol / L, KH2PO4 1.4mmol / L) or serum-free nutrient solution, added 2% maintenance solution, and incubated at 37°C with CO2 After 2 to 3 days, observe under a microscope, and when the cytopathic effect (CPE) reaches 85%, freeze at -20°C until use.

[0042] Step 2 Concentration and purification of virus

[0043]Freeze and thaw the collected virus repeatedly 3 times, centrifuge the suspension at...

Embodiment 2

[0061] The establishment of embodiment 2 hybridoma cell lines

[0062] Step 1 Mouse Immunization

[0063] Immunize 3 BALB / C mice (6-8 weeks old, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army) with 30%-45% of the purified JE full poison in Example 1: i Immunization for the first time: Japanese encephalitis virus 150 μg / mouse, add an equal volume of Fred's complete adjuvant (CFA, sigma company) and mix well, then inject BALB / C mice with 1ml / mouse subcutaneously in multiple points, 0.2ml / point, with an interval of 3 weeks. ii Second immunization: the dose and route are the same as above, this time with an equal volume of incomplete Freund's adjuvant (IFA, sigma company), with an interval of 3 weeks. iii. The third immunization: the dose is the same as above, without adjuvant, and the same volume of normal saline is used instead, and BALB / C mice are injected intraperitoneally; 10 days later, the eyeball b...

Embodiment 3

[0088] Example 3 In vivo induced ascites method to prepare monoclonal antibody

[0089] Ten BALB / C mice aged 6-8 weeks were pre-sensitized by intraperitoneal injection of 0.4ml liquid paraffin, and 7 days later, hybridoma cells 2B4 (CGMCC No.5124) were injected at 1×10 6 The above-mentioned presensitized mice were injected intraperitoneally with an amount of each (diluted with equal amount of normal saline to 500 μl / only), and the mice were observed after 10-14 days. For ascites, use a syringe to collect the ascites into a centrifuge tube, centrifuge at 2000r / min for 10 minutes, take the supernatant, which is the ascites with a large amount of monoclonal antibody 2B4, store it in a 4°C refrigerator for backup, and freeze it at -20°C or -40°C for long-term storage.

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Abstract

The invention belongs to the field of biology, and relates to a monoclonal antibody for resisting the Japanese encephalitis virus (JEV) and application thereof. The hybridoma cell strain 2B4 which can excrete the monoclonal antibody for resisting the JEV is preserved in the China General Microbiological Culture Collection Center on August 3, 2011, and the preservation number is CGMCC No. 5124. The monoclonal antibody for resisting the JEV is produced by the hybridoma cell strain 2B4 with the preservation number of CGMCC No. 5124. The monoclonal antibody can specifically identify the JEV, and both cell and animal experiments show that the monoclonal antibody has neutralizing activity and can be applied in the preparation of detecting reagents for detecting the JEV. The successful preparation of the anti-JEV monoclonal antibody lays a foundation for the further research on the JEV, the establishment of a faster and more sensitive and convenient detection method and the development of related diagnostic reagent kits, test paper and vaccines.

Description

technical field [0001] The invention belongs to the biological field and relates to a monoclonal antibody against Japanese encephalitis virus and its application. Background technique [0002] Japanese encephalitis virus (JEV), belonging to the Flaviviridae genus Flavivirus, is an enveloped single-stranded positive-sense RNA virus with three structural proteins: C, M and E, divided into four genotypes (Type I, II, III, IV). Culex tritaeniorhynchus is the main vector and is mainly prevalent in Asia. The main epidemic genotypes I and III in my country are one of the most important arboviruses that endanger people's lives and animal husbandry in China, and can cause severe neurological symptoms. , the fatality rate is as high as about 30%, and another 50% of patients will leave permanent neurological sequelae. According to statistics, the number of Japanese encephalitis cases in Asia reaches about 16,000 cases each year, and about 5,000 cases die. In China, except Xinjiang, Ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577C12R1/91
Inventor 周斌邓文蕾曹瑞兵苏小东陈溥言
Owner NANJING AGRICULTURAL UNIVERSITY
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