Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – Characterization and in vitro testing

Abciximab-grafted ultrasound sensitive microbubbles were developed for the diagnosis of stroke. The antibody fragment abciximab, which binds to the GP IIb/IIIa and αvβ3 receptors expressed by activated platelets, was chosen because most ischemic strokes are due to arterial thrombi that are mainly composed of platelets. The abciximab antibody fragment was activated by reduction of the disulfide bond for grafting on the microbubbles. The suspension was freeze-dried after the grafting was performed directly on the formed microbubbles. Quantification of the amounts of abciximab present on the surface of the microbubbles was assessed semi-quantitatively by flow cytometry, and quantitatively using radio-labeled abciximab. A protocol for human and rat platelets deposition and fixation was implemented and the expression of the GP IIb/IIIa receptor was validated by immunostaining. The abciximab-grafted microbubbles showed high static and dynamic binding to fixed platelets. Detection by ultrasonography of microbubbles bound on white and red clots gave higher signals compared to SonoVue microbubbles.