Figure 1
From: Rapid production of antigen-specific monoclonal antibodies from a variety of animals
Flowchart summarizing the generation of antigen-specific monoclonal antibodies (mAbs) from a variety of animals. (A) Fluorescence-activated cell sorting (FACS)-based antigen-specific plasma/plasmablast cell (ASPC) isolation from a variety of animals. Lymphocytes were stained with fluorescently labeled antibodies against IgG, ER-tracker and fluorescently labeled antigen. ASPCs defined as IgGlow endoplasmic reticulum (ER)high antigen+ were single-cell sorted into individual wells of 96-well plates. (B) Amplification of cognate pairs of VH and VL genes from single-cell-sorted ASPCs. Preparation of 3'-end homopolymer-tailed cDNA from single-cell-sorted ASPCs was performed automatically by MAGrahd. Cognate pairs of VH and VL genes were amplified by 5' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR). (C) Expression of recombinant mAbs. The amplified VH and VL genes were selectively assembled with IgH and IgL DNA cassettes by target-selective joint PCR (TS-jPCR) to generate linear IgH and IgL genes. The pairs of IgH and IgL genes were cotransfected into 293FT cells grown in 96-well culture dishes.