A real-time in vitro assay for studying functional characteristics of target-specific ultrasound contrast agents

To develop an in vitro assay for studying the feasibility of specific targeting of ultrasound contrast agents (USCAs) for ultrasound diagnostics by employing the parallel plate flow chamber, which provides an environment that mimics some aspects of the in vivo conditions like shear rate and flow effects.

USCAs based on air-filled microparticles (MP) were functionalized with specific antibodies using carbodiimide coupling chemistry and characterized by fluorescence activated cell sorter (FACS). The binding experiments were done by subjecting the MP to shear stress as they interact with the target-coated surface of the flow chamber.

A successive modification of MP with antibody and the glass surface with antigen was achieved and quantified. The binding studies showed specific attachment of targeted MP to EDB-FN (EDB domain of fibronectin) surface. The binding of MP via nonspecific interactions was minimal. The binding efficiency of antibody-loaded MP is dependent on the applied shear stress. An increase in the wall shear stress resulted in a decrease in binding efficiency. Binding efficiency was found to be correlated with the antibody density and antigen density on the interacting surfaces.

The results indicate that the test system developed is reliable for characterizing targeted MP without any additional labeling and can be used as a functionality assay for studying the binding characteristic of USCA with respect to different parameters like density of targeting antibodies on the microparticle surface and of target protein. In addition, the microparticles can be studied in detail under different shear rates and flow conditions. Further studies concerning the in vitro-in vivo correlation will be necessary to further increase the value of this in vitro method.