The Screening And Preparation Of Ebola Virus Glycoprotein Monoclonal Antibody

BackgroundThe Ebola virus disease is one kind of fatal hemorrhagic fever caused by the Ebola virus. By 2013, a total of 2387 people was infected,1310 patients died. The case fatality rate reaches to 54.9%. In 2014, the EBOV broke out again and swift to west Africa. This disease was no more limited in Africa. The United States, Britain and Spain have Ebola cases continuously. By December 8th,2015, the case number reaches to 28634.Among them,11314 patients died. The case fatality rate remains 39.5%.Nearly forty years have passed since the first outbreak of EBOV, there is still no specific treatment and vaccine available for clinical use. Ebola virus disease has become an international major communicable disease.The rapid, effective diagnosis of patients is important. The early isolation of patients is infective to reduce communication with each other. In this way,the disease can be well controlled.Classic Ebola virus infection diagnosis is relied on quantitative PCR technology, but it does not apply to all regions. This method need equipments and professional technical personnel. Rapid, sensitive, simple immunological diagnosis can help identify patients, control the source of infection. It is critical to prevention and control of epidemic diseases. Now we lack of this immune diagnostic products.There is no specific treatment to deal with Ebola virus disease. GP is very important to the invasion of Ebola virus. At the same time, GP is the main target protein to the diagnosis and treatment of Ebola virus. Although ZMapp targeted to GP has been made to deal with Ebola virus infection, but the treatment effect is still limited and incomplete. Thus developing a new kind of Ebola monoclonal antibody was in need. Providing more alternatives for the Ebola virus disease treatment can greatly increase the possibility to cure Ebola virus disease.Aim1)Screen and prepare high title of monoclonal antibodies against Ebola GP protein.2) Validation the antibody in the experiments conclude ELISA, Western Blot, Cell Immunofluorescence, Cell Immunocytochemistry.3) Complete the humanized antibodyMethodsThrough searching, screening antibody epitopes, synthetic Ebola GP protein polypeptide. Peptides combine with the cytokine immune adjuvant mice was injected to mouse. Through five weeks of immune method, prepare of immune spleen lymphocytes. Infuse spleen lymphocytes with SP2O to make the hybridoma. Through testing and screening, to get the hybridoma screting monoclonal antibody against GP protein.Using mice celiac injection of hybridoma to prepare monoclonal antibodies against Ebola GP protein.Complete purification and identification of the antibody. Complete monoclonal antibody subtype identification and sequencing. Complete antibody sensitivity and specificity detection. A false virus from Military academy of medical sciences expressing EBOV GP was used to construct the cell model. Validate the monoclonal antibody in the experiments concluding ELISA, Western blot, cell immunofluorescence test and the cellular immune chemistry experiment.Through genetic engineering technology, humanized monoclonal antibodies wasResults1) We successfully got two kinds of monoclonal antibodies named ZJEB8-01, ZJEDO-022) ZJEB8-01, ZJEDO-02 have the sensitivity reaches to 1:10000. They can be widely used in ELISA, Western Blot, Cell Immunofluorescence, Cell Immunocytochemistry.3) Humanized ZJEB8-01 was got.ConclusionWe successfully got two high title monoclonal antibody named ZJEB8-01, ZJEDO-02. They can be used in the detection of the Ebola virus disease. ZJEB8-01, ZJEDO-02 have the sensitivity reaches to 1:10000. They can be widely used in ELISA, Western Blot, Cell Immunofluorescence, Cell Immunocytochemistry. Humanized ZJEB8-01 has the ability to be used as alternative antibody drug for the treatment of the Ebola virus disease.