| Phage-displayed antibody library technology has the advantages of large capacity and great diversity,and has been applied to immunization therapies,which may lead to the development of new tools used for cancer diseases and diagnostic antibody screening.DNA sequences in phage-displayed native antibody library are mostly from IgM without stimulation.IgM-specific antibodies are typically of relatively low affinity in vivo compared to antibodies from the IgG isotype.After the assembly of the obtained scFv sequence to the full length of IgG,the affinity of antibody is usually 10-7 to 10-8 M,which does not meet the demand for high affinity antibody.This study f℃used on different selection approaches and then tuned the selection strategy accordingly to maximally sample the repertoire,according to different structural characteristics of antigens and project requirements.First,antigens were selected from surface extracellular domain protein,multiple transmembrane protein,human antigen and mouse antigen.Direct and indirect Elisa experiments were used to optimize antibody detection.Secondly,different forms of panning were selected including solid phase panning,liquid phase panning,cell based panning and high throughput instrument;and different initial concentration of antigen were selected to optimize panning method.Output antibody sequence number and positive rate were chosen to compare the optimal panning method.Thirdly,different forms of Elisa,buffer types and reagent concentrations were chosen to enlarge the detection window of assay method which improved positive values and reduced false positives rates.With series of optimizations,a number of high affinity antibodies(10-9 to 10-10 M)were obtained.This study on optimization of panning and screening methods showed improvement of the positive rates and antibody diversity of final antibody,which provided a methodological reference for high throughput selection of phage-displayed antibody library. |
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