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Assay Principle, Validation& Applications
HitHunter® cAMP Assays forBiologics and Small Molecules
Split β-Galactosidase Enzyme
Enzyme Fragment Complementation (EFC)Technology
2
Enzyme Fragment Complementation (EFC) technology consists of a β-galactosidase (β-gal) enzyme splitinto two inactive components, the enzyme donor peptide (ED) and enzyme acceptor (EA). Whenbrought together, ED complements with EA forming an active β-gal complex. With the addition ofsubstrate, the active β-gal catalyzes the formation of chemiluminescent products providing a highlyamplified signal.
Hydrolysis
Inactive Fragments
EFC-based cAMP Assay Principle
• At higher levels of cellular cAMP, the anti-cAMP antibody becomessaturated allowing the ED-cAMP complex to complement with the β-gal acceptor (EA) and form an active enzyme
• Following substrate addition, the active enzyme produces a signalthat is directly proportional to the cellular cAMP levels
Key Features and Benefits
• Flexible – Use with small molecules or biologics• Simple – Easy-to-follow protocol• Validated – In 400+ peer reviewed publications• Qualified – Unmatched performance for a variety of
applications• Robust – Exquisite sensitivity with large assay windows• Reliable – Reproducible lot-after-lot• Universal – Detect with any standard plate reader
Drug Discovery Applications forSmall Molecules and Biologics
Basic Research and AssayDevelopmentIdentify and fully characterizemolecules for correctpharmacology
Basic Research and AssayDevelopmentIdentify and fully characterizemolecules for correctpharmacology
ScreeningPerform functional cAMPresponse screening
ScreeningPerform functional cAMPresponse screening
Lead OptimizationProfiling molecules (rank order;EC50/IC50); obtain expectedpharmacology
Lead OptimizationProfiling molecules (rank order;EC50/IC50); obtain expectedpharmacology
Clinical StudiesMeasure drug efficacy andpotency in clinical samples
Clinical StudiesMeasure drug efficacy andpotency in clinical samples
QC BioassayRun quality control assays(potency assays for lotrelease and stability testing)
QC BioassayRun quality control assays(potency assays for lotrelease and stability testing)
Immunogenicity StudiesDetect neutralizingantibodies
Immunogenicity StudiesDetect neutralizingantibodies
Additional biologics applications
Rank Molecular Potency(EC50 or IC50 Comparison)
Profiling experiments of ranked ligands (agonists orantagonists) show assays can obtain the expectedpharmacology as indicated in the literature
Formeterol Isoproterenol Clenbuterol Salmeterol Salbutamol
EC50 1.7x10-10 1.7x10-9 2.7x10-9 2.8x10-9 1.6x10-7
S/B 14.5 11.6 9.1 13.1 12.3
Haloperidol Risperidon Chlorpromazine Clozapine
EC50 1.8x10-8 2.6x10-8 3.9x10-7 3.8x10-6
S/B 15.8 10.1 13.1 12.4
Measure Complex Assay Formats(Antagonists; Gαi-coupled Receptors)
Large assay windowsallow easier antagonistidentification andrescue of many difficultGαi-coupled receptors
Antagonist 1 Antagonist 2
EC50 2.4x10-8 8.0x10-9
S/B 4.2 6.2
Easily Identify Allosteric Modulators(PAMs and NAMs)
Quantify allosteric modulator activity by performing adose response curve study to determine the EC50and Schild analysis to show curve shifting withincreasing modulator concentrations
PAM Study on GRM4 (Glutamate Receptor) using cAMP Detection
Detect Inverse Agonism
Use assays to revealinverse agonists
Data from: Yao et.al; British Journal ofPharmacology (2006) 149, 145–154.
Inverse Agonist (SR144528) Studyon Cannabinoid Receptor CB2 using
cAMP Detection
Characterize Biologics
Characterization of theprotein SDF1α(stromal cell-derivedfactor 1α) using aCXCR4 Gαi-coupledchemokine receptorcell line
EC50 8.0x10-10
S/B 6.5
BiologicsExample
Obtain Excellent Reproducibility
Evaluate lot-to-lotconsistency forquality controlanalysis
Lot 1 Lot 2 Lot 3
EC50 8.7x10-11 7.4x10-11 7.6x10-11
S/B 10.2 12.0 12.6
BiologicsExample
Recognize Partial Agonists
• The large dynamic range of the assay helps to identify partialagonists
• Using the assay and validated cAMP Hunter™ cell lines showpartial agonism without difficulty
Data from: Yao et.al; British Journal ofPharmacology (2006) 149, 145–154.
BottomTopLogEC50HillSlopeEC50
Glucagon31.62514.8-7.9682.3401.076e-008
[des-His1,Glu9]-Glucagon26.65283.6-6.4671.9393.413e-007
Glucagon (des-His1, Glu9)-Glucagon
EC50 1.1x10-8 3.4x10-7
S/B 14.8 9.2
BiologicsExample
Partial Agonist (GW405,833) Studyon Cannabinoid Receptor CB2 using
cAMP Detection
Detect Neutralizing Antibodies
Performimmunogenicitystudies using serumand plasmasamples
BiologicsExample
Data from: Ryding et al. Journal of Immunological Methods (2013).
Neutralization Assay Study to select theMinimal Drug Concentration using the HumanFollicle-Stimulating Hormone (FSH) Receptor
Identify PDE Inhibitors
Study cAMP dependentphosphodiesterases(PDE) – enzymes thatregulate the cellularcAMP and cGMP levelsby controlling theirrates of degradation
0.04 μMcAMP
0.12 μMcAMP
0.37 μMcAMP
EC50 1.1x10-6 1.0x10-6 2.8x10-6
Phosphodiesterase Assay
BottomTopLogEC50HillSlopeEC50
0.37 uM cAMP-57.8398.030.4436-0.89212.777
0.12 uM cAMP-0.439397.230.01422-2.2381.033
0.04 uM cAMP5.45692.840.02689-2.4831.064
Quantify Low Levels of cAMP(Endogenous Receptor Studies)
Quantify endogenous(native) receptoractivity by monitoringlow levels of cAMP
EC50 1.8x10-7
S/B 12.3Best-fit valuesBottomTopLogEC50HillSlopeEC50
1321 N1 Gs PTGDR
86.25739.9-6.2621.6155.466e-007
Naked 1321 N1
47.09632.9-6.7360.95101.837e-007
Broad Range of Sample Compatibility
• Easily determine ligand activity in the presence of serum andplasma
• Assay works well with live cells or cell membrane fractions
2 μg/well
1 μg/well
0.5 μg/well
0.25 μg/well
EC50 7.5x10-7 8.4x10-7 1.2x10-6 2.5x10-6
BottomTopLogEC50HillSlopeEC50
2 g/well309.7496.7-6.1261.1917.475e-007
1 g/well308.6540.8-6.0750.90878.421e-007
0.5 g/well206.0474.8-5.9160.93331.213e-006
0.25 g/well132.8421.3-5.5960.75292.534e-006
0% FBS 5% FBS 10% FBS 25% FBS 50% FBS
EC50 1.70x10-8 1.57x10-8 1.59x10-8 1.90x10-8 2.28x10-8Best-fit valuesBottomTopLogEC50HillSlopeEC50
0% FBS
57.441813-7.7691.2031.703e-008
5% FBS
46.051997-7.8041.0491.571e-008
10% FBS
45.461954-7.7991.0521.588e-008
25% FBS
11.191878-7.7220.73121.898e-008
50% FBS
36.711525-7.6420.89322.281e-008
Simple Protocol
• Rapid, homogeneous protocol- Simply add cAMP assay reagents to stimulated cells,
incubate and read- Detection within 4 hours- Chemiluminescent readout using any standard plate reader
Scalable Assay(Miniaturization Compatible)
96-well 384-well 1536-well 3456-well
Reduce sample size and reagent costs
Weber M. (Merck), ELRIG 2006(384 and 3456 well format)
(384-well format)(1536-well format)
Validated Results
• Well-established, validated results since 2000- ~400 customer publications to date
HitHunter® cAMP Assay select citations
Simple User Manual
• Easy-to-follow, step-by-stepprocess
• Quick start guide• Helpful imagery, call-outs,
and schematics
Convenient Packaging
3 Sizes available for each applications
Bulk quantities are available upon request
Application Specific Kits
HitHunter cAMP Assay for Biologic HitHunter cAMP Assay for Small Molecules
Product No. Size Configuration Product No. Size Configuration
90-0075LM2 200/800 dp (96-well/384-well) 90-0075SM2 200/800 dp (96-well/384-well)
90-0075LM10 1,000/4,000 dp (96-well/384-well) 90-0075SM10 1,000/4,000 dp (96-well/384-well)
90-0075LM25 2,500/10,000 dp (96-well/384-well) 90-0075SM25 2,500/10,000 dp (96-well/384-well)
Complete Offering of Our EFC Technology
• Stable Cell Lines- Frozen cells (unlimited
culture)- Validated culture reagents
• Assay Ready Kits- Single-use frozen cells- Detection reagents- Cell plating media and
components
• LeadHunter™ Services- Leverage our expertise- Multiple flexible options- High throughput capabilities
• Custom Assay Development- Access to all technologies- Expertise from our R&D team- Flexible milestone structures- Experience with 300+ projects
cAMP Hunter™Stable Cell-lines
HitHunter® cAMP AssayDetection Reagents cAMP Hunter™ eXpress Kit
Assay Ready Kits withCells plus
Detection Reagents
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