APPLICATION NOTE

Quantification of anti-mTNF-alpha 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 are
lysed 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).
Monoclonal anti-TNF-alpha inhibitors are one of the most commons types of antibody drugs on themarket. The mechanism by which these drugs act is most often blocking of soluble TNF-alpha or theTNF receptor. Since TNF-alpha is produced through cleavage of a membrane bound form of the protein,
there is a risk that anti-TNF-alpha antibodies bind the membrane bound TNF-alpha and trigger effector
functions, such as ADCC. By using the iLite ADCC mTNF-alpha Activity Assay, the risk of an anti-TNFalpha antibody drug inducing ADCC can be quantified.

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 membrane bound TNF-alpha (iLite® mTNF-alpha (+) Target Assay ReadyCells). iLite® ADCC Effector (V) Assay Ready Cells also contain the Renilla Luciferase (RL) reporter
gene, under the control of a constitutive promoter, that allows anti-TNF drug-induced FL activity to benormalized with respect to the constitutive expression of RL. This renders assay results independent ofvariations in cell number, serum matrix effects, or lysis of the effector cells by the target cells. In addition,
we also describe the use of a negative control in the form of a target cell line without expression ofmembrane bound TNF-alpha (iLite® mTNF-alpha (-) 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 Infliximab 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

SwedenSwedenLABEL-DOC-0403, 4.0
Page 1/ 5

APPLICATION NOTE
Material and equipment needed
Material and equipment

Suggested supplier

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

Svar Life ScienceSvar Life Science
Svar Life Science
Gibco

BM5001
BM5013
BM501461870 (RPMI)
26140-079 (FBS)
15140-122(PenicillinStreptomycin)

Infliximab or analogues
Firefly/Renilla luciferase substrate

Janssen Biotech
PromegaNA
E2940, Dual-Glo®
Luciferase Assay
SystemPlate; 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 ofrecommended
suppliers
NA
NA
NA

NANA
NA
NA
NA

NA
NA
NANA

®

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

Preparation of calibrators (example given with infliximab)
The ADCC effect of the infliximab antibody from Janssen Biotech has successfully been measured incombination with a mix of ADCC Effector (V) Assay Ready Cells and mTNF-alpha (+) Target Assay
Ready Cells. As a negative control, a combination of ADCC Effector (V) Assay Ready Cells and mTNFalpha (-) Target Assay Ready Cells was used. In the present assay an Effector: Target ratio of 4:1 hasbeen used. The optimal ratio is dependent on the antibody and target cells used and should bedetermined each time a new assay is set up. The table below shows recommended dilutions of Infliximab
when making an 8-point calibration curve.

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-0403, 4.0
Page 2/ 5

APPLICATION NOTE

Relative light units

Calibrator12345678Figure 1. Example of Infliximab 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.

Infliximab
Calibrator solution conc.
(ng/mL)
1 000333
111
37124.1
1.4
0Table 1. Suggested calibrator solution concentrations for Infliximab.

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 0-500 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 mTNF-alpha (+)
Target Assay Ready Cells and mTNF-alpha (-) 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 mTNF-alpha (+)
Target Assay Ready Cells with 3.44 mL Diluent. The total volume of the diluted ADCCEffector (V) /Target mTNF-alpha (+) 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 ofthe mTNF-alpha (-) Target Assay Ready Cells with 860 µL Diluent. The total volume of thediluted ADCC Effector (V)/Target mTNF-alpha (-) 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.
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.

1The color of the cell suspension might vary from salmon pink to a more yellowish red. The difference in color
is due to small variations in pH of the solution and do not affect function of the cells.
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-0403, 4.0
Page 3/ 5

APPLICATION NOTE
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 concentrations ofInfliximab.
B: Measurement of the Renilla (RL) signal fornormalization, from the identical assay as in A.
C: Dose response curve after normalization of the specific
Firefly signal with the according Renilla signal.

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

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-0403, 4.0
Page 4/ 5

APPLICATION NOTE
AB and any attempt to reproduce the delivered iLite® Assay Ready Cells is an infringement of thesepatents.

QUICK GUIDE
Quantification of anti-mTNF-alpha ADCC activity usingiLite® ADCC Assay Ready Cells

1
Sample dilution•Equilibrate reagents and samples to room temperature – do not thaw cells andsubstrate 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 mTNF-alpha (+) Target
Assay Ready Cells, and a vial of mTNF-alpha (-) Target Assay Ready Cells in a 37Cwater bath. Mix the cell suspensions with a pipette in order to ensure ahomogeneous distribution of cells.
•Prepare diluted cell mix of ADCC Effector (V)/Target mTNF-alpha (+) Assay ReadyCells, and ADCC Effector (V)/Target mTNF-alpha (-) 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.

2Incubation4h3•Equilibrate the plate to room temperature•Prepare the Firefly luciferase substrate according to the supplier's instructions andadd 80 µL per well. Mix. Protect the plate from light. After 10 min incubation read ina luminometer.
•If appropriate, prepare the Renilla luciferase substrate according to the supplier'sinstructions and add 80 µL per well. Mix. Protect the plate from light. After 10 min
incubation read in a luminometer.

Read plate

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

SwedenSwedenLABEL-DOC-0403, 4.0
Page 5/ 5