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Data Analysis
From Assay Guidance Wiki
As with other functional assays, concentration response data may be fitted using a four-parameter logistic equation with variable slope to determine half maximal responses. Keep in mind that GTPγS assays will often show some degree of receptor reserve even though typically less than a cAMP or Ca++ mobilization assay and for this reason agonist EC50’s may not agree with Ki values for agonists determined in radioligand binding assays. For antagonists, Kb values may be determined from rightward curve shifts in the presence of a fixed antagonist concentration or from antagonist concentration response curves run at a fixed agonist concentration (at or somewhat below the concentration that produces a maximal response). For curve shift at a single antagonist concentration the following equation may be used to determine the Kb:
where EC50a is the agonist EC50 in the absence of antagonist and EC50b is that in the presence of antagonist.
For antagonist concentration responses the following equation is used (14):
where n is the slope of the agonist curve.
In antagonist concentration response experiments it is desirable to determine the agonist EC50 in each experiment along with the IC50 for the antagonist. Figure 8 below illustrates the use of both methods for measuring antagonist Kb values.
Figure 8: Examples of determining antagonist Kb values in GTPγS assays with a single antagonist concentration
