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Preparation Of Anti-rabies Virus Monoclonal Antibody And Establishment Of Colloidal Gold Kit

Posted on:2020-05-18Degree:MasterType:Thesis
Country:ChinaCandidate:X F HeFull Text:PDF
GTID:2370330596974275Subject:Biology
Abstract/Summary:PDF Full Text Request
Research purposes:In order to explore the expression conditions of rabies virus monoclonal antibodies,provide experience for the subsequent in-depth study of preparation of stable,high titer and good quality antibodies,and establish a colloidal gold kit for the detection of rabies virus,providing a simple,fast and sensitive detection method for rabies virus.So this paper conducted a preliminary study on the preparation of monoclonal antibodies of rabies virus and colloidal gold kit.In this paper,hybridoma cells as the research object,the conditions of hybridoma cells in vitro culture,preparation of rabies virus monoclonal antibody,purification and screening of Anti-rabies Virus Hybridoma Cells,the conditions of expanded cultivat-ion by bioreactor and establishment of colloidal gold kit were studied.Results:1.Under the condition of static culture,the hybridoma cells observed with microscope adherent growed to the wall of bottle,the surface of that were smooth and translucent,rounded,tended to aggregate and grow,and the growth rate was fast;After domestication with low serum concentration,the cells grew similarly in 2%,5%and 10%serum concentration,but could not grow normally in serum-free condition..Cells could grow normally when initial culture density was 2.5×10~5/mL,but could not grow normally when the initial culture density was less than 1×10~5/mL.Cells could grow in 1640 and DF12 media,but could not grow in DMEM and F12 media;The secretion of antibodies by cell lines was unstable and degenerated.2.The mice injected with the two cell lines were positive for ascites by in vivo induction,and the antibody titer was 1:1600;Mycoplasma contamination was found in the 69 and 102 cell lines detected by PCR.3.Four hybrid cell lines of B6,D10,G6 and C9 were obtained by monoclonal screening.;The bioreactor failed to culture hybridoma cells,and the cells could not grow in the bioreactor.4.The prepared colloidal gold was red and stable.After testing,there was no agglomeration and precipitation.;The preliminary experiment showed that the protein can label colloidal gold.;The minimum concentration of monoclonal antibody reacted with colloidal gold was 200 times dilution.;The ELISA optimization result was,when coated with 69 mouse antibody,the ascites dilution factor was 1:100,the antigen dilution factor was 1:100,and the 102 antibody dilution factor was 1:100,the OD value was the highest,being 0.733.When coated with 102 mouse peritoneal antibodies,the ascites dilution factor was 1:100,the antigen dilution ratio was 1:100and 1:1000,and the 69 antibody dilution factorw were 1:10 and 1:100,the OD The highest valuew were 0.792 and 0.784 respectively.The gold standard test strip was dripped with samples after assembly,quality control line and detection line marking.The positive samples had red precipitation lines in both the quality control line and the detection line,while the negative samples had only precipitation lines in the detection line,and there was no reaction in the quality control line.Conclusion:By studying the monoclonal antibody of rabies virus and the establishment of the gold standard kit,the conditions of in vitro culture of hybridoma cells were obtained;The rabies virus monoclonal antibody was successfully prepared by in vivo induction method;and four hybrids were obtained.A colloidal gold kit for detecting rabies virus was initially established.In the experiment,unstable antibody secretion and mycoplasma contamination appeared in cell lines,which affected the preparation of monoclonal antibody.In the subsequent experiments,we need to rethink and improve it.
Keywords/Search Tags:rabies virus, monoclonal antibody, Hybridoma cell, Colloidal gold kit
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