Cell based assays are required for the detection of
biological activity and hence are required for use as
potency assays, detection of neutralizing antibodies and detection and quantification of the
effector cell function of therapeutic antibodies or the quantification of the
potency or
neutralizing antibody response to
virus vectors such as AAV vectors used in
gene therapy.
Cell-based
assay are also required for the quantification of
antibody mediated
effector functions including complement-dependent
cytotoxicity (CDC),
antibody-dependent cellular
phagocytosis (ADCP), and
antibody-dependent
cell-mediated
cytotoxicity (ADCC).
Cell based assays are difficult to adapt for use on automated immuno-
assay platforms. Reporter-genes that
encode readily visible proteins that can be quantified using
fluorescence or
luminescence such as
luciferases (firefly, Renilla, Gaussia, Nano
Luciferase etc), fluorescent proteins such as
green fluorescent protein (GFP) or dsRED or an
enzyme such as
chloramphenicol acetyltransferase (CAT) or a
protease and respond to
signal transduction, that is directly related to the
mechanism of action of a
drug, can be used to quantify the
potency of a
drug following binding of a
drug to a soluble target or
cell surface target (
receptor or other cell-surface molecule), anti-drug neutralizing antibodies, and
therapeutic antibody induced
effector cell function. The principal of the invention is that reporter-
gene product or by-product produced either during the course of the cell-based
assay or onconclusion of the
cell based assay is quantified either in the cell medium or cell supernatant, for a secreted
protein, or following
lysis of the cells with a suitable passive
lysis buffer. The reporter-
gene product such as firefly
luciferase is then detected in the cell medium or cell supernatant or cell lysate using an antibody pair (
monoclonal or polyclonal) specific for the
gene product such as a firefly
luciferase labelled with the dual detection
system specific for an ELISA or a particular automated assay platform such as
Meso Scale Discovery electro-
chemiluminescence (MSD-ECL), Luminex, SMC, Alpco, AlphaLISA, Gyros or
label free detection using SPR such as the Biacore platform. The expression of the reporter-
gene product such as firefly
luciferase can be normalized with respect to the expression of a second
reporter gene product such as
Renilla luciferase or Nano
Luciferase under the control of a constitutive
promoter.