TY - JOUR
T1 - Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists
AU - Lallemand, Christophe
AU - Kavrochorianou, Nadia
AU - Steenholdt, Casper
AU - Bendtzen, Klaus
AU - Ainsworth, Mark A
AU - Meritet, Jean-Francois
AU - Blanchard, Brigitte
AU - Lebon, Pierre
AU - Taylor, Peter
AU - Charles, Peter
AU - Alzabin, Saba
AU - Tovey, Michael G
N1 - Copyright © 2011 Elsevier B.V. All rights reserved.
PY - 2011
Y1 - 2011
N2 - A cell-based assay has been developed for the quantification of the activity of TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.
AB - A cell-based assay has been developed for the quantification of the activity of TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.
U2 - http://dx.doi.org/10.1016/j.jim.2011.08.022
DO - http://dx.doi.org/10.1016/j.jim.2011.08.022
M3 - Journal article
VL - 373
SP - 229
EP - 239
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -