Antibody Methods and Protocols

Abstract

The generation of recombinantly produced fl uorescent antibody derivatives that are derived from full-length immunoglobulin G (IgG) has until now been problematic. One major reason for that lies in different and partially incompatible secretion- and folding-requirements of antibodies and green fl uorescent protein (GFP) derived fl uorescent entities in mammalian cells. The use of citrine as fl uorescent fusion entity can overcome this limitation. Citrine is a modi fi ed yellow fl uorescent protein (YFP) derivative which in contrast to GFP and yellow fl uorescent protein (YFP) folds effectively and properly in the endoplasmic reticulum (ER) of mammalian cells. Provided that proper design parameters regarding fusion positions and linker/connector sequences are applied, citrine can be fused to different positions of IgGs and be expressed without interfering with secretion capability or functionality of IgG–citrine derivatives. Because IgG– citrine fusions are stable and retain biophysical properties of IgGs, they can be expressed and puri fi ed in the same manner as regular antibodies. IgG–citrine fusions not only retain the binding properties (af fi nity and speci fi city) of antibodies but also contain Fc-regions (useful for immunoassay applications), and are fully de fi ned molecules (in contrast to antibody conjugates with fl uorophores).