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Hybridoma cell strain ZJEB8-01, Ebola-virus GP albumen resistant monoclonal antibody, and preparation and application of Ebola-virus GP albumen resistant monoclonal antibody

A hybridoma cell line, Ebola virus technology, applied in the direction of antiviral immunoglobulin, biochemical equipment and methods, material inspection products, etc., can solve the problems of inaccuracy, limited therapeutic effect, lack of drugs, etc., and achieve high Effects of Specificity and Sensitivity

Active Publication Date: 2015-11-25
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the drug ZMapp, which is a mixture of three antibodies, has been developed internationally, the therapeutic effect is still limited and uncertain
If the antibody lacks effectiveness and safety in the clinical phase I, then people will face another situation where there is no drug available, and even lack of available drugs

Method used

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  • Hybridoma cell strain ZJEB8-01, Ebola-virus GP albumen resistant monoclonal antibody, and preparation and application of Ebola-virus GP albumen resistant monoclonal antibody
  • Hybridoma cell strain ZJEB8-01, Ebola-virus GP albumen resistant monoclonal antibody, and preparation and application of Ebola-virus GP albumen resistant monoclonal antibody
  • Hybridoma cell strain ZJEB8-01, Ebola-virus GP albumen resistant monoclonal antibody, and preparation and application of Ebola-virus GP albumen resistant monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of the monoclonal antibody of embodiment 1 anti-Ebola GP protein

[0036] (1) Immunization of mice: BALB / C mice aged 6 weeks were selected for immunization. For the first immunization, the 20 peptide of the Ebola virus GP protein (Ebola virus GP protein No. 412-431) cross-linked KLH Amino acid sequence: DNDSTASDTPATTAAGPLK) and QuickAntibody-mouse5W were mixed uniformly at a volume ratio of 1:1. Each BALB / C mouse 0.1ml (20 peptide 100ug of Ebola GP protein), intramuscularly injected into the inner thigh. On the 21st day, a dose of booster immunization was given in the same way. On the 35th day, a small amount of tail blood was collected for ELISA determination, and the antibody titer reached 1:32000, followed by a booster immunization by tail vein injection, and cell fusion was carried out 3 days later.

[0037] Among them, the 20 peptide of Ebola virus GP protein was synthesized by solid phase method, carried out on the peptide synthesizer (431A) of ABI...

Embodiment 2

[0045] Embodiment 2 carries out the identification (qualitative) detection of Ebola virus GP protein with this monoclonal antibody

[0046] The anti-Ebola GP protein monoclonal antibody prepared by embodiment 1 can be used for identification (qualitative detection), and the identification method can be realized by the following methods:

[0047] Western Blotting: The Ebola adenovirus recombinant vaccine loaded with Ebola virus GP protein was cultured in HEK-293 cells, and the cultured cell lysate was used for Western Blotting detection with the monoclonal antibody, and the target band appeared at the corresponding position, indicating that the detection to the expression of the Ebola virus GP protein. Specific steps:

[0048] (1) SDS polyacrylamide gel electrophoresis: For the method, refer to F. Osper et al., "A Guide to Molecular Biology Experiments" (Science Press, 1998). Using 10% separating gel and 5% stacking gel, the electrophoresis condition is a voltage of 150V, and...

Embodiment 3

[0052] Embodiment 3 monoclonal antibody detects the sensitivity of Ebola virus GP protein

[0053] The anti-Ebola virus GP protein monoclonal antibody prepared in Example 1 can be used for quantitative detection of various surface The level of the Ebola virus GP protein that reaches can be achieved by the following methods:

[0054] Indirect ELISA method:

[0055] ① Properly dilute the test sample in 0.01M pH9.6 carbonate buffer coating solution, 100ul / well, incubate at room temperature for 2h or coat overnight at 4°C.

[0056] ②Empty the liquid and pat dry the residual liquid, wash the plate three times with 0.01MpH7.4PBS-Tween20 washing solution.

[0057] ③ Block with 2% BSA-0.01M PBS at pH 7.2 for 1 hour.

[0058] ④ Wash the plate 3 times as above.

[0059] ⑤Add 1:100 diluted monoclonal antibody, 0.1ml / well, and react at room temperature for 2 hours.

[0060] ⑥After washing the plate 3 times as above, add goat anti-mouse IgM-HRP, 0.1ml / well, and react at room temperat...

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Abstract

The invention relates to a hybridoma cell strain ZJEB8-01, an Ebola-virus GP albumen resistant monoclonal antibody, and preparation and application of the Ebola-virus GP albumen resistant monoclonal antibody. The hybridoma cell strain ZJEB8-01 can be used for secreting the Ebola-virus GP albumen resistant monoclonal antibody. Binding of the monoclonal antibody and the 412th-431st amino acid sequence antigen peptides of Ebola-virus GP albumen has high specificity and sensitiveness, and the monoclonal antibody can be applied to preparation of Ebola-virus GP albumen detection reagents. Sequencing of the monoclonal antibody is completed, and the material basis is laid for humanization transformation of monoclonal antibody sequences, research and development of the neutralizing antibody for neutralizing Ebola viruses and development of the antibody into clinical Ebola virus treatment antibody in later period.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell line ZJEB8-01, an anti-Ebola virus GP protein monoclonal antibody and its preparation and application. Background technique [0002] Ebola virus disease is a fatal hemorrhagic fever caused by Ebola virus infection. In 1976, the Ebola virus broke out for the first time in Zaire, and then broke out in Central Africa on a small scale. As of 2013, a total of 2,387 people became ill and 1,310 died, with a case fatality rate of 54.9%. In 2014, the Ebola virus broke out again, sweeping across West Africa and across continents. Ebola cases occurred in the United States, Britain, and Spain one after another. As of May 27, 2015, there were 26,969 cases and 11,135 deaths, and the case fatality rate was still as high as 41.3%. Nearly 40 years have passed since the first outbreak of Ebola, and there is still no specific treatment or vaccine available for clinical use in the wor...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/68G01N33/577G01N33/569
Inventor 姚航平吴晓鑫吴南屏李兰娟
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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