APPLICATION NOTE

Detection of C5aR inhibitory activity usingiLite® C5a 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
Complement component 5a (C5a) is a 74 amino acid small protein fragment of complement protein 5(C5). C5 is cleaved to C5a and C5b by C5 convertase enzymes as a result of complement activation.
But C5 can also be cleaved by factors of the coagulation and fibrinolytic pathways. (1) C5a is a highly
potent anaphylatoxin and chemoattracting peptide, with the ability to increase blood vessel permeability,
stimulate cytokine release from myeloid cells, and expression of adhesion molecules on endothelialcells. (2)Main pro-inflammatory effector functions are induced by C5a binding to the G protein-coupled seventransmembrane-containing receptor, C5aR1 (CD88). Rapidly after cleavage of C5 into C5a and C5b,
C5a is metabolized by carboxypeptidases which removes the C-terminal arginine and forms C5adesArg, a less potent ligand of C5aR1. C5a can also bind to a second receptor, C5aR2 (C5L2 orGPR77), however the biological effects associated with C5a binding C5aR2 are less well understood,
both anti-inflammatory and pro-inflammatory effects have been described in literature. The C5areceptors are expressed in a wide range of cells and tissues, and the effect of C5a activation isdependent on the location of the C5a receptors. (3, 4)
While the complement system is an important part of the innate immune defense against pathogens,research has shown that excessive complement activation including C5a-C5aR interaction plays acentral role in several autoimmune and neurodegenerative disorders as well as in acute and chronic
inflammatory conditions. Given the strong evidence of a pathogenic role for C5a-C5aR in many
conditions both ligand and receptor are promising therapeutic targets. Clinical studies of pharmacologic
candidates inhibiting C5a and C5aR1 are ongoing. (2,3,5)
The iLite® platform offers a cell-based assay that enables the studies of C5a, receptor C5aR1 andtheir interaction.

Principle of the assay
The iLite® C5a Assay Ready Cells are engineered cells, optimized to express Firefly luciferase (FL)under the control of a C5a responsive promoter and exhibits a dose-dependent increase in firefly
luciferase (FL) reporter gene activity following treatment of cells with increasing concentrations of humanC5a. The cells also contain the Renilla luciferase (RL) internal standardization gene under the controlof a constitutive promoter that renders assay results independent of cell number and provides a meansfor correcting for cytotoxic effects that may be encountered with some biological samples. Some
increase in RL reporter-gene activity may be observed, however, at high non-physiologic concentrationsof C5a and consequently normalization of C5a-induced FL activity relative to the expression of RL
activity is not recommended for routine use. Normalization of C5a-induced FL activity relative to theexpression of RL activity remains, however, a valuable means of correcting for sample cytotoxicity.
The luciferase signals can be measured in a luminometer following addition and incubation of luciferase
substrate.

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 NOTEThe Firefly luciferase signal is proportional to the functional activity of C5a in the sample. In the presenceof inhibitory activity against C5aR, the functional activity of the present C5a is reduced, resulting in adecreased stimulation of Firefly luciferase production. Thus, the Firefly luciferase signal is inversely
proportional to the amount of inhibitory activity against C5aR in a sample (fig 1). The iLite® C5a Assay
Ready Cells can therefore be utilized as an assay for detection of C5aR inhibitor activity in test samples,
including human serum.

Material and equipment needed
Material and equipment

Suggested supplier

ReferenceiLite® C5a Assay Ready Cells
Diluent (DMEM containing 9% heat inactivated FBS +1% Penicillin-Streptomycin).

Svar Life Science
Gibco

C5aR antagonist W-54011
C5a or analogues
Firefly/Renilla luciferase substrate

Tocris Bioscience
R&D Systems
Promega

Plate; White walled micro well plate suitable forluminescence
Microplate Luminometer with appropriate reading
software – no filter on luminometer

PerkinElmer

BM407531966-021 (DMEM)
26140-079 (FBS)
15140-122 (PenicillinStreptomycin)
5455
2037-C5
E2920, Dual-Glo
Luciferase Assay System
6005680

Incubator, 37 C with 5% CO2
Water bath, 37 C
Single-channel and multi-channel pipettes with
polypropylene disposable tips
Polypropylene tubes or plate for dilution
Single-use polypropylene reservoir
Plate shakerTimerContact Svar Life
Science for list ofrecommended suppliers
NANA
NANA

NANA
NANANA
NA
NA
NA

NANA
NAProtocol
Preparation of C5a receptor inhibitor
The C5aR antagonist W-54011 from Tocris Bioscience has successfully been used to neutralize thefunctional effect of C5a induced Firefly luciferase expression in iLite® C5a Assay Ready Cells (refer tothe table and graph at page 3)
Assay preparation and incubation
1. Design a plate layout. It is recommended to perform the test at least in duplicates.
2. Perform a serial dilution of the reference C5aR inhibitor. Ensure matrix consistency between
reference solutions, control solutions, and sample solutions.
3. Add 20 µL of the reference C5aR inhibitor dilutions, controls, and samples to assigned wells(final concentration will be a quarter of solution concentration).
4. Thaw the vial of iLite® C5a Assay Ready Cells in a 37 C water bath with gentle agitation. Thecell suspension is mixed very carefully ten times with pipette to ensure a homogeneous
distribution of cells.
5. Dilute 250 µL cell suspension with 5.75 mL Diluent.
6. Add 40 µL diluted cells to each well.
7. Place the lid on the plate, mix and incubate the plate for 30 minutes at 37 C with 5% CO2.
8. Add 20 µL of 16 ng/ml C5a to all wells (final concentration will be 4 ng/mL C5a).
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
9. Place the lid on the plate, mix and incubate for 5 hours at 37 C with 5% CO2.
Final4 ng/mL C5aFigure 1. Example of C5aR inhibitory curve

W-54011123

Suggested calibrator solution
concentrations, nM
1600
800
400

45620010050782513910116.3
3.1
1.6

120

Table 1. Suggested calibrator solution concentrations for
C5aR inhibitor

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

Precautions






This application note is intended for professional laboratory research use only. The data andresults 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 thesupplier’s/manufacturer’s instructions or product specifications accompanying the individualmaterial and instruments.
Dispose of all sample specimens, infected or potentially infected material in accordance withgood microbiological practice. All such materials should be handled and disposed as thoughpotentially 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.

Propriety 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

SwedenSweden

LABEL-DOC-0511, 1.0
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APPLICATION NOTEQUICK GUIDE
Detection of C5aR inhibitory activity using iLite® C5a Assay Ready Cells

1Sampledilution• Equilibrate reagents and samples to room temperature – do not thaw cells and substrate reagents at thisstage• Dilute reference C5aR inhibitor, controls and samples.
• Add 20 µL of ref. C5a inhibitor solutions, controls and diluted samples to pre-assigned wells.
• Thaw the cell vial in a 37 °C water bath. Mix cell suspension with a pipette to ensure a uniform cellsuspension. Dilute the cells• Add 40 µL diluted cells to each well.

• Incubate at 37 °C with 5% CO2 for 30 min.
2Incubation
30 min3Add C5a• Add 20 µL C5a to each well.

• Incubate at 37 °C with 5% CO2 for 5 hours.
4Incubation
5h5
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 a luminometer.

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

References
1. Manthey et al. Complement Component 5a (C5a). Int J Biochem Cell Biol. 2009
Nov;41(11):2114-7.
2. Woodruff et al. Inhibiting the C5–C5a receptor axis. Mol Immunol. 2011 Aug;48(14):1631-42.
3. P N Monk et al. Function, Structure and Therapeutic Potential of Complement C5a Receptors.
Br J Pharmacol 2007 Oct;152(4):429-48.
4. Colley et al. Structure and characterization of a high affinity C5a monoclonal antibody thatblocks binding to C5aR1 and C5aR2 receptors. MAbs. 2018 Jan;10(1):104-117.
5. Hawksworth et al. New concepts on the therapeutic control of complement anaphylatoxin
receptors. Mol Immunol. 2017 Sept;89:36-43.

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-0511, 1.0Page 4/ 4