cAMP Product Solutions

Homogeneous cAMP Detection Assay Solutions for Small Molecules and Biologics

cAMP cell-based assays measure cAMP levels in cells as a direct indication of GPCR functional status (activation or inhibition). This direct measurement is obtained by quantifying cellular cAMP accumulation levels in a dose-dependent manner with cell-based assays that are homogeneous and scalable.

 

Eurofins DiscoverX provides robust, easy-to-use, and high throughput screening (HTS) HitHunter® cAMP cell-based assays that accurately detect cellular cAMP levels in a variety of applications without the need for wash steps, optimization, or specialized equipment. These assays provide a simple, validated detection system, optimized with over 125 cAMP Hunter™ cell lines, and include large signal-to-background, gain-of-signal responses. They can be used to monitor GPCR activation or inhibition with cAMP Hunter, ChemiSCREEN™, or non-Eurofins DiscoverX cell lines. Easily obtain reproducible results with high sensitivity, rank order ligands based on molecular potency, or determine pharmacological profiles of your ligands.

 

Used in the screening of millions of data points and referenced in 100s of peer reviewed publications ranging from basic research to clinical applications, these assays provide accurate pharmacology of your ligand or small molecule and biologics while giving you the flexibility to work with multiple cell lines.

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cAMP Product Highlights

  • Optional Formats – Select from 100s of stable cell lines, frozen ready-to-assay cells, and assay ready eXpress and bioassay kits for chemiluminescent or fluorescent assay detection
  • Assay Ready Kits – Complete, ultra-convenient kits with optimized reagents for over 125 cAMP Hunter™ cell lines for monitoring GPCR activation using small molecules or biologics
  • High Throughput – Simple, rapid, and scalable assays that accurately detect cellular cAMP levels in 96-well through 3456-well formats
  • Validated Applications – Referenced products in over 400 peer reviewed publications ranging from basic research to clinical applications

 

 

Advantages of cAMP Product Solutions

Optimized for a Variety of Applications
  • Perform functional screens using small molecules or biologics (antibodies, peptides)
  • Determine pharmacological profiles and rank order ligands
  • Detect neutralizing antibodies (immunogenicity studies using serum and plasma)
  • Characterize phosphodiesterase inhibitors

HitHunter cAMP Assays

  • Eliminate optimization steps with an assay that has been designed and qualified for use with over 125 cAMP Hunter cell lines
  • Achieve precise characterization of ligand pharmacology with large assay windows, sensitive detection, and wide dynamic range for ligand bias studies and many more applications
  • Reproducible assay performance without fluorescence or serum interference and no need for specialized equipment

cAMP Hunter™ Cell Lines and Assay Ready Kits

  • Over 125 stable cell lines with unlimited culture and overexpressed naturally coupled, wild-type GPCRs
  • Ultra-convenient, complete eXpress kits for quick assessment without the need for cell culture
  • Ready-to-use bioassay kits provide cryopreserved cells and optimized reagents with high lot-to-lot reproducibility to support the lifetime of the biologic drug

ChemiSCREEN Cell Lines

  • Cell lines containing a promiscuous G protein, Gα15, to help funnel signaling to a common calcium readout, regardless of G protein coupling status
  • High functional expression of receptors with the ability to assay both agonist and antagonist in a single well
  • Stable cell lines for continuous culture and increased reliability and reproducibility assay results
  • Perform orthogonal assay confirmation such as second messenger signaling, e.g. cAMP, and ERK phosphorylation, for most targets

ChemiBRITE Cell Lines

  • Cell lines modified to flash with added brightness upon GPCR activation
  • Exceptional high signal-to-background ratio with no assay interference with autofluorescent compounds
  • Tool box capabilities allows generation of your own stable cell lines

ChemiSCREEN and ChemiBRITE Frozen Cells

  • Ready-to-Assay GPCR frozen cells come with plating media and enough cells to run either 96- or 384-well plate assays in full or half plate formats
  • Qualified for cAMP and calcium second messenger assays with results within 24 hours to accelerate your data generation
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Products

Detection Kits

  • HitHunter cAMP Assays – Accurately detect cellular cAMP levels using biologics or small molecules ligands in a variety of applications. These Enzyme Fragment Complementation (EFC) based assays monitor GPCR activation through chemiluminescent detection of cAMP production in cAMP Hunter™ cell lines as well as non Eurofins DiscoverX cell lines.

Assay Ready eXpress and Bioassay Kits

  • cAMP Hunter eXpress and bioassay kits – Complete assay ready kits with specific cell line, optimized reagents, and plates for quick EFC-based chemiluminescent detection. Bioassays include additional optimization for high lot-to-lot reproducibility.

Cell Lines and Ready-to-Assay Frozen Cells

  • cAMP Hunter cell lines – Naturally coupled, stable cell lines qualified to use with chemiluminescent EFC-based HitHunter® cAMP Assays, AssayComplete™ cell culture reagents, and control ligands
  • ChemiSCREEN stable cell lines, parental cell lines, and ready-to-assay frozen cells – Cells based on our proprietary Chem-1 and related host cell backgrounds that contain an endogenous expression of Gα15. Gα15 is a promiscuous G protein to help funnel signaling to a common calcium readout, regardless of G protein coupling status. Use cell lines or frozen cells with chemiluminescent or fluorescent assays, and parental cell lines to generate your own GPCR stable cell lines. Additional second messenger signaling, e.g. calcium mobilization, and ERK phosphorylation, for most targets can be used to perform orthogonal assay confirmation.
  • ChemiBRITE stable cell lines and ready-to-assay frozen cells – Cells engineered to flash with added brightness upon GPCR activation and developed to provide the greatest readout flexibility, including luminescent and fluorescent calcium flux as well as cAMP. These cells express a proprietary mutant version of clytin, a calcium activated photoprotein.

Custom Products

  • Custom Capabilities – Custom cell lines, assays, and protein development capabilities optimized to your requirements

HitHunter cAMP Assay Principle

HitHunter cAMP Assay Principle

 

The cAMP G protein-dependent pathway involves a heterotrimeric (α/β/γ) G-protein containing a GDP molecule bound to the Gα subunit, which holds the trimer together. Upon activation, GDP is exchanged for GTP, leading to the dissociation of the Gβ/Gγ dimer from Gα. Both parts remain anchored to the membrane and become free to act upon their downstream effectors and initiate unique intracellular signaling responses. The activated Gα subunit interacts with and regulates many effector molecules such adenylyl cyclase that can ultimately lead to the accumulation of cAMP (a second messenger).

 

HitHunter cAMP assays are competitive immunoassays amenable for high-throughput and utilizes the Enzyme Fragment Complementation (EFC) technology where a fragment ß-galactosidase (ß-gal) enzyme donor (ED) is conjugated with cAMP. This ED-cAMP conjugate and cellular cAMP compete for binding to an anti-cAMP antibody (Ab). With low levels of cellular cAMP, most of the ED-cAMP binds to the cAMP Ab, making the ED-cAMP unable to complement with the enzyme acceptor (EA). With high levels of cellular cAMP, the anti-cAMP antibody becomes saturated allowing the ED-cAMP complex to complement with the ß-gal acceptor (EA) and form an active enzyme. The active enzyme then subsequently hydrolyzes a substrate to produce a chemiluminescent signal that is directly proportional to the amount of cAMP in the cells.

Assay Workflow

Assay Workflow

 

HitHunter cAMP Assays are cell-based functional, immunoassays with a chemiluminescent readout. The assays are available as complete kits that are robust, highly sensitive, and easy-to-use to study GPCR activity through cAMP production. The kits contain all the reagents needed for the detection of cAMP from whole cells expressing Gαi- and Gαs-coupled receptors induced with a biologic or small molecule ligand. The flexible assay system has been designed to work in agonist or antagonist mode for 96- and 384-well plate formats. After plating and stimulation of cells, the user simply adds the HitHunter cAMP Assay reagents to the cell following the homogeneous, simple protocol provided.

Accurately Rank Molecular Potency of Ligands

Accurately Rank Molecular Potency of Ligands

HitHunter cAMP assays provide powerful tools for drug discovery screening and revealing of the correct ligand rank order and pharmacological profile. Profiling experiment using a cAMP Hunter CHO-K1 adrenergic receptor β2 cell line to test five agonists. The data highlights the receptor’s sensitivity to the various agonists, ultimately revealing the correct rank order of the agonists and showing the agonist salbutamol (EC50 of 160 nM) is less potent than the control ligand isoproterenol (EC50 of 1.7 nM).

Easily Determine Pharmacological Profiles of Complex Assay Formats

Easily Determine Pharmacological Profiles of Complex Assay Formats

Large assay windows and sensitive detection makes HitHunter cAMP assays ideal for studying complex assay formats like Gαi-coupled receptor antagonists. Comparative study of two antagonists of the Gαi-coupled metabotropic glutamate receptor 2 (GRM2) using a cAMP Hunter CHO-K1 GRM2 cell line. Results indicate that the Antagonist 2 (IC50 of 8 nM) is a more potent inhibitor compared to Antagonist 1 (IC50 of 24 nM).

Obtain Excellent Reproducibility and High Sensitivity for Testing Biologics

Obtain Excellent Reproducibility and High Sensitivity for Testing Biologics

Perform quality control assays, including potency assays for lot release and stability testing in biologics. A. Evaluation of lot-to-lot consistency using a GLP agonist, GLP-1(7-36), and the cAMP Hunter Gαs-coupled GLP-1R bioassay that incorporates the HitHunter cAMP Assay for Biologics. Results indicate the assay's excellent reproducibility for all three lots tested with S:B ratios over 10 fold and EC50’s ranging from only 76 pM to 86 pM. B. Evaluation of lot-to-lot consistency using a biologic ligand, SDF1α, and the cAMP Hunter Gαi-coupled CXCR4 receptor (chemokine C-X-C motif receptor 4) cell line. Results show high sensitivity detection and excellent reproducibility with overlapping S:B ratios of ~6 and EC50’s ranging from only 689 pM to 796 pM.

Identify Allosteric Modulators, Partial Agonists, Inverse Agonists, and Silent Agonists

Identify Allosteric Modulators, Partial Agonists, Inverse Agonists, and Silent Agonists

HitHunter cAMP assays’ superior assay performance, with its large assay window and wide dynamic range, allows for easy identification of a diverse set of pharmacological ligands such as positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), partial agonists, and more. Easily identify partial agonists. This experiment uses a cAMP Hunter Gαs-coupled glucagon receptor (GCGR) CHO-K1 cell line to analyze two agonists. Results reveal [des-His1, Glu9]- glucagon exhibits partial agonism (S:B of 9.2; EC50 of 340 nM) compared to the full native agonist, glucagon (S:B of 14.8; EC50 of 11 nM).