Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

Christophe Lallemand; Nadia Kavrochorianou; Casper Steenholdt; Klaus Bendtzen; Mark A Ainsworth; Jean-Francois Meritet; Brigitte Blanchard; Pierre Lebon; Peter Taylor; Peter Charles; Saba Alzabin; Michael G Tovey

doi:10.1016/j.jim.2011.08.022

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

J Immunol Methods. 2011 Oct 28;373(1-2):229-39. doi: 10.1016/j.jim.2011.08.022. Epub 2011 Sep 3.

Authors

Christophe Lallemand¹, Nadia Kavrochorianou, Casper Steenholdt, Klaus Bendtzen, Mark A Ainsworth, Jean-Francois Meritet, Brigitte Blanchard, Pierre Lebon, Peter Taylor, Peter Charles, Saba Alzabin, Michael G Tovey

Affiliation

¹ Laboratory of Biotechnology and Applied Pharmacology, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Cachan, France.

PMID: 21910993
DOI: 10.1016/j.jim.2011.08.022

Abstract

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB 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 NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adalimumab
Animals
Antibodies, Monoclonal / blood
Antibodies, Monoclonal / immunology
Antibodies, Monoclonal / pharmacology*
Antibodies, Monoclonal, Humanized / blood
Antibodies, Monoclonal, Humanized / immunology
Antibodies, Monoclonal, Humanized / pharmacology
Antibodies, Neutralizing / analysis*
Antibodies, Neutralizing / blood
Antibodies, Neutralizing / immunology
Arthritis, Rheumatoid / blood
Arthritis, Rheumatoid / drug therapy
Crohn Disease / blood
Crohn Disease / drug therapy
Culture Media / chemistry
Culture Media / pharmacology
Dose-Response Relationship, Drug
Gene Expression Regulation, Enzymologic / drug effects
Humans
Infliximab
K562 Cells
Luciferases / genetics
Luciferases / metabolism*
Luciferases, Firefly / genetics
Luciferases, Firefly / metabolism
Luciferases, Renilla / genetics
Luciferases, Renilla / metabolism
NF-kappa B / genetics
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Regulatory Sequences, Nucleic Acid / genetics
Serum / chemistry
Serum / immunology
Tumor Necrosis Factor-alpha / antagonists & inhibitors*
Tumor Necrosis Factor-alpha / immunology
Tumor Necrosis Factor-alpha / pharmacology

Substances

Antibodies, Monoclonal
Antibodies, Monoclonal, Humanized
Antibodies, Neutralizing
Culture Media
NF-kappa B
Recombinant Fusion Proteins
Tumor Necrosis Factor-alpha
Infliximab
Luciferases
Luciferases, Renilla
Luciferases, Firefly
Adalimumab