H. Gazzanosantoro et al., A NONRADIOACTIVE COMPLEMENT-DEPENDENT CYTOTOXICITY ASSAY FOR ANTI-CD20 MONOCLONAL-ANTIBODY, Journal of immunological methods, 202(2), 1997, pp. 163-171
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.