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Preparation Of Monoclonal Antibody For Type ? Poliovirus

Posted on:2019-05-28Degree:MasterType:Thesis
Country:ChinaCandidate:Y H TangFull Text:PDF
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Poliovirus?PV?is the pathogen of polio in the world.It mainly causes acute flaccid paralysis by invading the patient's central nervous system.Since 1988,when the world health organization?WHO?proposed the global polio eradication initiative in 2000,the number of polio cases has decreased by more than 99%,from an estimated 350,000 cases to 416 cases reported in 2013.The number of polio-endemic countries has decreased from 125 in 1988 to 2 in 2017?Pakistan,Afghanistan?,and the world is on the edge of eradicating polio.Poliovirus monoclonal antibodies,which could be used for epidemiological surveillance,poliovirus vaccine immunization level assessment,derived polio case studies,have an important significance.The main content of this study is to discuss the preparation of monoclonal antibody for type I poliovirus.Vero cells were used to successfully proliferate the poliovirus Sabin strain I.The virus was identified by the VP1 gene with the reverse transcriptional amplified viral RNA and the identified in HuBei Provincial Center for Disease Control and Prevention.Calculated by using the Reed-Muench formula,the titration of the virus was 108.217 TCID50/0.1mL.At the same time,the titer of the poliovirus Sabin type I,II and III was determined respectively,and the completely CPE was found within 4 to 5days after the inoculation of virus in cells.One liter of poliovirus-cell suspension was collected,and concentrated with PEG.Next,the virus was purified by sucrose density gradient centrifugation,virus was determined with SDS-PAGE gel electrophoresis of the virus structural protein VP1,VP2 and VP3,purified viral protein content was measured as 2.764 mg/mL.The purified poliovirus type Sabin strain I was used to immunize once every two weeks and the fourth booster immunization conducted to SPF grade Balb/c mice?6 to8 weeks old,female?.Four batches of mice were immunized.Square matrix titration test was used to determine the optimal antigen concentration,in the establishment of indirect antibody ELISA method,half of the mouse serum immune titer reached more than 6,400.Three days after the 4th immunization,splenic lymphocytes was obtained from the immunized mice,and fused with myeloma cells?SP2/0-Ag14?via PEG,the cell fusion rate was 15.63%,3.12%,66.49%,33.33%,46.65%and 14.21%respectively.Eight positive hybridoma cells were screened by indirect ELISA.One stable hybridoma cell line,named G8G7 were established by a limited dilution method.Monoclonal antibodies prepared by cell culture,which by indirect ELISA assay for antibody titer is 1/8,was measured to be 1/58 of neutralization antibody titer by neutralization test.
Keywords/Search Tags:monoclonal antibody preparation, poliovirus, vero cell lines
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