HTRF Resource Library

HTRF® cAMP assays: discussion of best practices for assay design and data analysis

By: N. Burford

Bristol-Myers Squibb

Abstract:

HTRF cAMP assays generate a ratiometric signal which can then be transformed to a concentration of cAMP by using a cAMP standard curve.  When the x-axis of the standard curve is plotted on a logarithmic scale a sigmoidal curve is generated which can be fit to a 4-parameter curve fit using Ymin., Ymax., Slope, and EC₅₀.  The ratiometric signal for the unknown sample can then be transformed to a concentration of cAMP by determining x from y using the standard curve generated equation.  Ideally, the compound response should fall within what is often mistakenly termed the “linear range” of the standard curve for sensitive detection and transformation of raw data into a concentration of cAMP generated.  This presentation will discuss best practices for assay design to obtain high quality data. The presentation will focus on the effect of data transformation of ratiometric data to cAMP data on concentration response curves for compounds which activate or inhibit adenylyl cyclase, with emphasis on EC₅₀/IC₅₀ and Ymax. changes that occur.  By understanding the effect that data transformation has on concentration response curves, a Gs-coupled allosteric potentiator assay will be described.