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Related Experiment Videos

Stable, sensitive, fluorescence-based method for detecting cAMP.

Jayne Hesley1, Janet Daijo, Anne T Ferguson

  • 1Molecular Devices, Sunnyvale, CA 94089, USA.

Biotechniques
|September 20, 2002
PubMed
Summary
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This study presents a sensitive, high-quality fluorescence immunoassay for measuring cyclic adenosine monophosphate (cAMP). This assay is fast, accurate, and suitable for high-throughput screening in drug discovery.

Area of Science:

  • Biochemistry
  • Cellular Biology
  • Pharmacology

Background:

  • Cyclic adenosine monophosphate (cAMP) is a crucial secondary messenger linking cell surface receptor signals to nuclear transcriptional changes.
  • Monitoring cAMP activity is vital for understanding cellular functions, human diseases, and advancing drug discovery.
  • Existing methods for cAMP detection may lack the sensitivity or throughput required for comprehensive analysis.

Purpose of the Study:

  • To evaluate the performance of a novel fluorescence-based competitive immunoassay for cAMP detection.
  • To determine the assay's sensitivity, accuracy, and suitability for high-throughput screening (HTS).
  • To validate the assay using cellular models stimulated with known cAMP-modulating agents.

Main Methods:

  • Development and optimization of a fluorescence-based competitive immunoassay in a 384-well microplate format.

Related Experiment Videos

  • Determination of assay sensitivity (detection limit) and quality (Z'-factor) using purified cAMP.
  • Treatment of HEK 293 cells with forskolin and isoproterenol to induce cAMP changes.
  • Measurement of dose-response curves and calculation of EC50 values.

    • Main Results:

    • The assay demonstrated a low detection limit of 0.1 nM cAMP.
    • A high Z'-factor (>0.83) indicated excellent assay quality and reliability.
    • Consistent results were observed regardless of reaction times between 10 and 60 minutes.
    • EC50 values for forskolin (11 µM) and isoproterenol (123 nM) in HEK 293 cells aligned with literature values.
    • The assay proved to be fast, accurate, and non-radioactive.

    Conclusions:

    • The developed fluorescence immunoassay is a sensitive and robust method for quantifying cAMP levels.
    • Its high throughput capability makes it ideal for drug discovery screening.
    • This assay provides a valuable tool for studying cAMP-mediated signaling pathways in various biological contexts.