Example 3
Binding to CD25 expressing Karpas 299 cells was examined by staining Karpas299 cells with test articles (anti-CD25 primary antibodies) starting at a concentration of 20 g/ml antibodies followed by semi-log dilution series (7-point) for 30 minutes on ice. This was followed by staining with a secondary antibody (Alexa Fluor 647-AffiniPure F(ab′)2 Fragment Rabbit Anti-Human IgG Fcγ fragment—(Jackson ImmunoResearch)) at a concentration of 1 μg/ml for 30 minutes on ice. All samples were stained in duplicates. Live cells were gated using FSC vs SSC parameters by flow cytometry during sample acquisition. Mean fluorescence intensity (MFI) of stained cells were plotted on an XY chart, graphing MFI against the log of the concentration, and the data fit to a non-linear regression curve from which the EC50 is calculated. Results are shown in
