Microbubble Construction E-selectin Mab PEG This slide shows the structure of E-selectin targeting phospholipid microbubble. Monoclonal antibody against mouse E-selectin is conjugated to the microbubble surface via streptavidin-biotin linkage (biotinylated antibody is joined to biotinylated phospholipid microbubble via a streptavidin bridge). Microbubbles are biotinylated by attaching biotin to the end of PEG (polyethylene glycol - an essential component of phospholipid microbubbles). This strategy has limitations: Streptavidin is a micro-organism (Streptomyces avidinii) protein and likely to cause unwanted immunogenic effects therefore best avoided for human applications. This conjugation strategy is non-site directed, where biotin is attached to the amine groups, the latter are distributed nearly uniformly throughout the surface topology of the antibody. Under these circumstances, the binding efficiency of targeting microbubbles may be suboptimal as the antibodies could be randomly orientated, and biotin-streptavidin molecules in close proximity to the antigen binding sites are likely to interfere with target binding. The use of whole monoclonal antibodies may cause non-specific binding and unwanted immunological effects due to the presence of the Fc region of conjugated whole antibodies. We propose as a solution to all these 3 issues, the use of maleimide linkage (much less or none immunogenic, maleimide containing antibodies have been tried in humans without significant side effects); it also allows conjugation of antibodies in a site-directed manner (linking the hinge-region sulphydryl groups of the antibodies to the microbubble phospholipid shell). This method can be applied for the conjugation of Fab’ (pronounced fab prime) fragments, the latter does not contain the Fc region of the antibody. Petros please note: However, in our exploratory stages of translational research in developing microbubble targeted imaging, the streptavidin-biotin linkage system is useful for five reasons: (a) biotinylated microbubbles can be stored for several months, ready for a quick one step conjugation to a variety of targeting agents when needed; (b) it forms a strong conjugation bond; (c) the antibody incorporation is relatively efficient (upto 50,000 antibodies per bubble) (d) the non-covalent conjugation chemistry requires less stringent conditions compared with the covalent maleimide linkage chemistry, allowing easy titration experiments to determine the optimal antibody:microbubble ratio for microbubble targeting; and (e) although streptavidin may cause unwanted immunogenic effects, it has not adversely affected microbubble targeting experiments involving mice. Other Ab conjugation strategies Maleimide linkage (site directed) Fab’ fragments instead of whole mAb Strepatvidin B Biotin Fig adapted from Takalkar et al