Establishment And Optimization Of EV71Humanized Specific Monoclonal Antibody Secretory Cells Screening Method

Humanized monoclonal antibodies are widely used in the prevention and controlof infectious diseases, cancer treatments and other areas. In order to obtain ahumanized monoclonal antibody, this study explores the specific antibody-secreting Bcells immortalized method and conditions; establishes specific B cells selectionmethod by using nano-beades coupled with antigen to screen HIV/P24and EV71/VP1humanized monoclonal antibodies. This study includes the following aspects:1) In order to screen HIV-1/P24monoclonal antibodies, express the recombinantplasmid pTWIN1-P24and purify HIV-1/P24antigen by affinity chromatography.2) Lymphocytes separated from collected HFMD (Hand-Foot-Mouth Disease)patients’ blood to gain peripheral blood mononuclear cells (PBMC). DetectEV71/VP1IgG of EV71positive samples by enzyme-linked immunosorbentassay (ELISA) and indirect immunofluorescence technique immunofluorescentantibody technique (IFA) and IgM by ELISA.3) Explore activation condition in vitro cell culture to prompte memory B cellssecreting antibodies. Add EBV, CPG and CyA to stimulate B lymphocytes beenimmortalized after recovering HFMD patients’ lymphocyte cells. Select B cellsby using CD19beads and detected antibody titer after fold dilution.4) Compare two different media coupled with antigen and establish cell-selectionmethod. Directly screen specific antibody secreting B cells by resin column andnano-beads coupled with antigen. PCR amplificated the heavy and light chaingene of specific B cells after fold dilution. Transfect to293T cells to secreteantibodies therefore obtain humanized monoclonal antibodies.