A NONRADIOACTIVE COMPLEMENT-DEPENDENT CYTOTOXICITY ASSAY FOR ANTI-CD20 MONOCLONAL-ANTIBODY

A simple and non-radioactive complement-dependent cytotoxicity assay w as developed to determine the relative potency of an anti-CD20 mAb, ID EC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear r elationship between the relative fluorescence unit generated and the n umber of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reprodu cible dose-response curves in the concentration range of 0.02-3.3 mu g /ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision a nd accuracy. Specificity was demonstrated by the lack of activity of i mmunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (g amma 4) which does not bind complement. The assay is able to detect de gradative changes in the molecule caused by heat, light and proteolyti c treatments, suggesting its use as a stability-indicating method. Fin ally, the Alamar blue method compared favorably with other more conven tional methods used to assess cell viability, The assay has the desire d properties for use as a potency assay for quality control testing of anti-CD20 mAb.