There are a growing number of antibody and small molecule therapeutic candidates and this demands a quick and efficient technique to screen for biomarkers that internalize effectively upon binding. FITC-Streptavidin-ZAP (FITC-SA-ZAP) provides for the efficient determination of internalization of cell surface biomarkers upon binding of antibodies or peptides. The construct that makes this method effective was formed by crosslinking a fluorescent reporter, in this case fluorescein (FITC) and Streptavidin (SA) to the ribosome-inactivating protein, saporin (ZAP).
The conjugate used in screening potential targeting agents or cells is a mixture of a biotinylated targeting agent mixed in a 1:1 molar ratio with FITC-labeled Streptavidinylated saporin (FITC-SA-ZAP). The bond between Streptavidin and biotin is rapid and essentially nonreversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. The method provides a definitive assay readout: fluorescence within 1 hour and cell death in 72 hours. This method is designed for rapid screening, in a quick and reproducible manner, for specificity and internalization in various cell types to explore suitability of targeting agents.
FITC-SA-ZAP recognizes cells targeted by biotinylated materials.