Humanized antibody plasmid DNA was modified to allow the distance between the Fc fragment and antigen binding sites of immunoglobulin G (IgG) antibodies to be studied. Specific variable heavy (VH) and variable light (VL) genes were inserted into heavy and light chain plasmids so that dye molecules can be easily attached to the expressed protein, and further inspection of antibody structure and function can be conducted via single molecule Förster resonance energy transfer (FRET). First, VH and VL genes were inserted into humanized antibody plasmids through the technique of ligation. The ligation product was then transformed into Escherichia coli cells, allowing the success of the ligation reaction to be determined by methods of agarose gel electrophoresis and sequencing of the plasmid DNA.
"Modifying Antibody DNA for Site-Specific Binding,"
DePaul Discoveries: Vol. 2
, Article 4.
Available at: http://via.library.depaul.edu/depaul-disc/vol2/iss1/4