Antibody Internalization
Confocal microscopy and FACS analysis are used to measure the internalization of the target receptor.
T0 min : Fluorescent labeled test antibody is added to the cells expressing the target antigen on surface. T60 min: The internalization of the test antibody is visualized and measured by the decrease of intensity of the fluorescent signals on the cell surface and the increased intensity of the fluorescent signals inside the cells. T4 hour: The internalization of the test antibody is evident with further decrease of intensity of the fluorescent signals on the cell surface and the increase in fluorescent signals localized in the endocytic vesicles.
RESULTS/DATA:
A. Quantitative Surface Binding: Relative Mean Fluorescence value was calculated to determine the binding affinity of test antibodies to the cell surface antigens. B. Internalization Measurement:Fluorescent labeled test antibody was added to the cells expressing target antigen at time 0 min. Test antibody internalization is evident from the capping and punctate staining of endocytic vesicles after incubation at 37C for 90 minutes.
FACS analysis is used to measure the internalization of target receptor after binding to the test antibody on cell surface. The target receptor is visualized by fluorescent labeling (black). After the test antibody bound to the receptor on the cell surface, the internalization of the receptor-antibody complex is measured by the decrease of fluorescent signal indicating the lower concentration of the receptor present on the cell surface (red).
Antibody Evolution, Engineering and GenerationBENEFITS:
Direct visual method that is sensitive, efficient, and reproducible
Rapid method with high sensitivity to compare and evaluate antibody internalization efficiencies
Aids selection of optimal monoclonal antibody-conjugate pairs