GraphPad Curve Fitting Guide

50% of what? Relative vs absolute IC50

50% of what? Relative vs absolute IC50

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50% of what? Relative vs absolute IC50

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IC50 can be ambiguous

50% of what? There can be more than one answer in some situations.

The concepts of IC50 is fundamental to pharmacology. The IC50 is the concentration of an inhibitor where the response (or binding) is reduced by half. Seems simple enough. But when you actually go to fit data to determine these values, there are several complexities and ambiguities.

The rest of this article is about IC50 (I for inhibition, for downward sloping dose-response curves). All the ideas can be applied to stimulatory curves and EC50 (E for effective) as well. Just stand on your head when you view the figures!.

The ideal situation

This figure shows an ideal situation:

The green symbols show measurements made with controls. The ones on the left (Blank) have no inhibitor, so define "100%". The ones on the right are in the presence of a maximal concentration of a standard inhibitor, so define "0%". The data of the experimental dose-response curve (red dots) extend all the way between the two control values.

When fitting this curve, you need to decide how to fit the top plateau of the curve. You have three choices:

Fit the data only, ignoring the Blank control values.

Average the Blank control values, and set the parameter Top to be a constant value equal to the mean of the blanks.

Enter the blank values as if they were part of the dose-response curve. Simply enter a low dose, perhaps 10-10 or 10-11.  You can't enter zero, because zero is not defined on a log scale.

The results will be very similar with any of these methods, because the data form a complete dose-response curve with a clear top plateau that is indistinguishable from the blank. I prefer the third method, as it analyzes all the data, but that is not a strong preference.

Similarly, there are three ways to deal with the bottom plateau: Fit the data only,  set Bottom to be a constant  equal to the average of the NS controls, and put the NS controls into the fit as if they were a very high concentration of inhibitor.

That is the ideal situation. There is no ambiguity about what IC50 means.

A potentially ambiguous situation

The graph below shows an unusual situation where the inhibition curve plateaus well above the control values (NS) defined by a high concentration of a standard drug. This leads to alternative definitions of IC50.

Clearly, a single value cannot summarize such a curve. You'd need at least two values, one to quantify the middle of the curve (the drug's potency) and one to quantify how low it gets (the drug's maximum effect).

The graph above shows two definitions of the IC50.

The relative IC50 is by far the most common definition, and the adjective relative is usually omitted.  It is the concentration required to bring the curve down to point  half way between the top and bottom plateaus of the curve. The NS values are totally ignored with this definition of IC50. This definition is the one upon which classical pharmacological analysis of agonist and antagonist interactions is based. With appropriate consideration of the biological system and concentrations of interacting ligands, estimated Kd values can often be derived from the IC50 value defined this way (not so for the "so-called absolute IC50" mentioned below).

The concentration that provokes a response halfway between the Blank and the NS value is sometimes called the absolute IC50,  The horizontal dotted lines show how 100% and 0% are defined, which then defines 50%.  This term is not entirely standard. Since this value does not quantify the potency of a drug, the authors of the International Union of Pharmacology Committee on Receptor Nomenclature (1)  think that the concept of absolute IC50 (and that term) is not useful (R. Neubig, personal communication). I agree.

The concept (but not the term "absolute IC50") is used to quantify drugs that slow cell growth. The abbreviation GI50 is used for what we call here the absolute IC50. It is also used by the Environmental Protection Agency (EPA) in evaluating endocrine disrupters (Appendix A). That document uses the term IC50 to refer to the absolute IC50, and the term EC50 to refer to the relative IC50. It doesn't use the terms relative and absolute.

If you really want to use the absolute IC50, the next page has instructions for fitting a curve to find it.


1. R. R. Neubig et al. International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol Rev (2003) vol. 55 (4) pp. 597-606

Download the Prism file used to create all the graphs in this article.