APPLICATION NOTE

Quantification of anti-EGFR ADCC activity
using iLite® ADCC Assay Ready Cells
For research and professional use only. Not for use in diagnostic procedures.
This application note contains a suggested protocol and performance data.
Each individual laboratory must set up their own method and perform relevant validations.

Background
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism whereby pathogenic cells arelysed by lymphocytes, most often Natural Killer (NK) cells. The mechanism involves binding ofantibodies to surface antigens on the pathogen. Crosslinking of these antibodies to NK cells through thebinding of the Fc-portion to Fc receptors on the NK cells leads to activation of the NK cell and formationof an immune synapse with the pathogenic cell. The NK cell releases cytotoxic granules containing
granzymes and perforin into the synapse, leading to apoptosis of the targeted cell (1).
Deficient signaling of the EGFR and other receptor tyrosine kinases in humans is associated withdiseases such as Alzheimer's, while over-expression is associated with the development of a widevariety of tumors. Interruption of EGFR signaling, either by blocking EGFR binding sites on theextracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can prevent thegrowth of EGFR-expressing tumors and improve the patient's condition. Therefore, many novel
therapeutic approaches are aimed at the EGFR, for example the monoclonal antibodies cetuximab andpanitumumab. Since the activity of these therapeutic antibodies is mediated in part by antibodydependent cell-mediated cytotoxicity (ADCC) there is a need to assess the ADCC activity of these
therapeutic antibodies, which can be performed with iLite® ADCC Assay Ready Cells.

Principle of the assay

The iLite® ADCC Assay Ready Cells are engineered cells that enable antibody-dependent cell-mediatedcytotoxicity (ADCC) to be examined through the specific expression of Firefly luciferase. When theantibodies of interest bind to the antigens on the surface of the target cell, the target-bound antibodies
will be presented to the Fc receptors (FcγRIIIa) on the effector cell. When the Fc-portion of the targetbound antibodies binds to the receptor, multiple cross-linking of the two cell types occurs. This will initiate
a signaling cascade which triggers the expression of Firefly luciferase (FL) in the effector cell. In thisapplication note, we describe the use of an effector cell line (iLite® ADCC Effector (V) Assay ReadyCells) that over-express FcγRIIIa and contain the FL reporter gene that responds to the principaltranscription factors that mediate signaling from the FcγRIIIa receptor, together with a positive target
cell line which over-expresses EGFR (iLite® EGFR (+) Target Assay Ready Cells). iLite® ADCC Effector(V) Assay Ready Cells also contain the Renilla Luciferase (RL) reporter gene, under the control of aconstitutive promoter, that allows drug-induced FL activity to be normalized with respect to theconstitutive expression of RL. This renders assay results independent of variations in cell number, serum
matrix effects, or lysis of the effector cells by the target cells. In addition, we also describe the use of anegative control in the form of a target cell line depleted of EGFR expression (iLite® EGFR (-) Target
Assay Ready Cells). The Firefly luciferase signal can be measured in a luminometer following addition
and incubation of luciferase substrate. The Firefly luciferase signal is proportional to the functional
activity of cetuximab in the sample (Fig 1).

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

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APPLICATION NOTE
Material and equipment needed
Material and equipment

Suggested supplier

ReferenceiLite ADCC Effector (V) Assay Ready Cells
iLite® EGFR (+) Target Assay Ready Cells
iLite® EGFR (-) Target Assay Ready Cells
Diluent (RPMI 1640 + 9% heat inactivated FBS+ 1% Penicillin Streptomycin)

Svar Life Science
Svar Life Science
Svar Life Science
Gibco

BM5001
BM5035
BM503661870 (RPMI)
26140-079 (FBS)
15140-122(PenicillinStreptomycin)

Cetuximab or analogues
Firefly/Renilla luciferase substrate

LillyPromegaNA
E2940, Dual-Glo®
Luciferase AssaySystemPlate; White walled micro well plate suitable forluminescence
Microplate Luminometer with appropriate
reading software – no filter on luminometer

PerkinElmer

6005680

Contact Svar Life Science
for list of recommended
suppliers

NAIncubator, 37C with 5% CO2
Water bath, 37C
Single-channel and multi-channel pipettes withpolypropylene disposable tips
Polypropylene tubes or plate for dilution
Single-use polypropylene reservoir
Plate shaker
Timer

NA
NA
NA

NA
NA
NA

NA
NA
NA
NA

NA
NA
NA
NA

®

Preparation of calibrators (cetuximab)
The ADCC effect of the cetuximab antibody from Roche has successfully been measured in combination
with a mix of ADCC Effector (V) Assay Ready Cells and EGFR (+) Target Assay Ready Cells. As anegative control, a combination of ADCC Effector (V) Assay Ready Cell and EGFR (-) Target Assay
Ready Cells was used. In the present assay an Effector: Target ratio of 4:1 has been used. The optimal
ratio is dependent on the antibody and target cells used and should be determined each time a new
assay is set up. The table below shows recommended dilutions of cetuximab when making an 8-pointcalibration curve.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

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LABEL-DOC-0407, 3.0
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APPLICATION NOTECetuximabCalibrator
12345678Figure 1. Example of cetuximab calibration curve using Firefly
Luciferase substrate Dual-Glo® Luciferase Reagent. Values areshown as mean of triplicate ± SD and values on x-axis are given as
final concentration in the wells before addition of Dual-Glo®
Luciferase Reagent.

Calibrator solution conc.
(ng/mL)
800267
133
6733
175.70Table 1. Suggested calibrator solutionconcentrations for cetuximab.

ProtocolAssay preparation and incubation
1. Design a plate layout.
2. Dilute calibrators, controls and samples to fall within the expected in assay values (= finalconcentration) of 0-400 ng/mL.
3. Add 40 µL calibrators, controls and samples in duplicate to assigned wells.
4. Thaw the vial of ADCC Effector (V) Assay Ready Cells and the vials of EGFR (+) Target Assay
Ready Cells and EGFR (-) Target Assay Ready Cells in a 37C water bath with gentle
agitation1.
5. Mix the cell suspensions very carefully at least 10 times with a pipette in order to ensure ahomogeneous distribution of cells.
6. Dilute 200 µL of the ADCC Effector (V) Assay Ready Cells and 200 µL the EGFR (+) Target
Assay Ready Cells with 3.44 mL Diluent. The total volume of the diluted ADCC Effector (V)/Target EGFR (+) Assay Ready Cells mixture is 3.84 mL.
7. In a separate tube, dilute 50 µL of the ADCC Effector (V) Assay Ready Cells with 50 µL of theEGFR (-) Target Assay Ready Cells with 860 µL Diluent. The total volume of the diluted ADCCEffector (V)/Target EGFR (-) Assay Ready Cells mixture is 960 µL.
8. Add 40 µL of the diluted cells to each well to be tested.
9. Place the lid on the plate and mix on a plate shaker at minimum of 750 rpm for 10 sec.
Alternatively, mix the cell suspensions very carefully in the wells by pipette. Insufficient mixing
can cause reduced assay sensitivity.
10. Incubate for 4 hours at 37°C with 5% CO2.

1

The color of the cell suspension might vary from salmon pink to a more yellowish red. The difference in coloris due to small variations in pH of the solution and do not affect function of the cells.
Svar Life Science AB
Lundavägen 151P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

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APPLICATION NOTE
Adding substrate solutions
11. Equilibrate the plate and the substrate solutions to room temperature.
12. Prepare the Firefly luciferase substrate in accordance with the supplier’s instructions andadd 80 µL per well. Mix and protect the plate from light. After 10 minutes incubation at room
temperature read in a luminometer.
13. If appropriate, prepare the Renilla luciferase substrate in accordance with the supplier’s
instructions and add 80 µL per well. Mix and protect the plate from light. After 10 minutes
incubation at room temperature read in a luminometer.

Normalization
The reporter gene used for normalization, Renilla luciferase, is under the control of a tyrosine kinase
promoter, and is thus constitutively expressed. Unspecific effects such as serum matrix effects ordifferences in cell number can be obviated by relating the specific Firefly signal with the Renilla
normalization signal through simple division.

ABCRatio (FL/RL)

Figure 2.
A: Measurement of the specific Firefly (FL) signal.
Cells were stimulated with increasing concentrationsof cetuximab.
B: Measurement of the Renilla (RL) signal fornormalization, from the identical assay as in A.
C: Dose response curve after normalization of thespecific Firefly signal with the according Renillasignal.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

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APPLICATION NOTE
PrecautionsThis application note is intended for professional laboratory research use only. The data and results
originating from following the Application Note should not be used either in diagnostic procedures
or in human therapeutic applications.
Use and handle the material and instruments referenced according to the suppliers'/manufacturers'
instructions or product specifications accompanying the individual material and instruments.
Dispose of all sample specimens, infected or potentially infected material in accordance with good
microbiological practice. All such materials should be handled and disposed as though potentially
infectious.
Residues of chemicals and preparations are generally considered as biohazardous waste andshould be inactivated prior to disposal by autoclaving or using bleach. All such materials should be
disposed of in accordance with established safety procedures.

Proprietary Information
In accepting delivery of iLite® Assay Ready Cells the recipient agrees not to sub-culture these cells,
attempt to sub-culture them or to give them to a third-party recipient, and only to use them directly inassays. iLite® cell-based products are covered by patents which are the property of Svar Life Science
AB and any attempt to reproduce the delivered iLite® Assay Ready Cells is an infringement of thesepatents.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0407, 3.0
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APPLICATION NOTEQUICK GUIDE
Quantification of anti-EGFR ADCC activity usingiLite® ADCC Assay Ready Cells

1Sample dilution• Equilibrate reagents and samples to room temperature – do not thaw cells and substrate
reagents at this stage• Dilute calibrators, controls and samples.
• Add 40 µL of calibrators, controls and diluted samples to pre-assigned wells.
• Thaw a vial of ADCC Effector (V) Assay Ready Cells, a vial of EGFR (+) Target Assay ReadyCells, and a vial of EGFR (-) Target Assay Ready Cells in a 37C water bath. Mix the cellsuspensions with a pipette in order to ensure a homogeneous distribution of cells.
• Prepare diluted cell mix of ADCC Effector (V)/Target EGFR (+) Assay Ready Cells, and ADCCEffector (V)/Target EGFR (-) Assay Ready Cells respectively.
• Add 40 µL diluted cell mixtures to each well and mix.

• Incubate at 37 °C with 5% CO2 for 4 hours.

2Incubation
4h

3
Read plate• Equilibrate the plate to room temperature• Prepare the Firefly luciferase substrate according to the supplier's instructions and add 80 µL
per well. Mix. Protect the plate from light. After 10 min incubation read in a luminometer.
• If appropriate, prepare the Renilla luciferase substrate according to the supplier's instructions
and add 80 µL per well. Mix. Protect the plate from light. After 10 min incubation read in aluminometer.

Troubleshooting and FAQ
Please consult the Svar Life Science website www.svarlifescience.com

References
1. Weiner GJ. Building better monoclonal antibody-based therapeutics. Nat Rev Cancer 15(6):361-70 (2015).

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSweden

LABEL-DOC-0407, 3.0
Page 6/ 6