Section15:Data Types and Associated Rules for Radioligand Binding Assays: Inhibition Mode

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Normalized Results

For radioligand binding methods, the use of Inhibition is recommended to quantify the ability of individual concentrations of a substance to inhibit the total specific binding of radioligand. The use of % bound for normalization is discouraged, but it’s recommended that biologists calculate and track changes to % bound as a measure of assay performance.

Calculation:

Derived Results: Absolute IC50 and Relative IC50

Absolute IC50: The molar concentration of a substance that reduces the specific binding of a radioligand to 50% of the maximum specific binding.

Relative IC50: The molar concentration of a substance that reduces the specific binding of a radioligand to 50% of the range of the binding curve (Top – Bottom) for that particular substance.

Notes:

• For incomplete curves, the response data must span 50% for an IC50 to be used for the determination of a Ki.
• The Top and Bottom parameters should be within +/- 20% of the Top and Bottom dynamic range control values.

Derived Results: Ki

The equilibrium dissociation constant of a test compound (Ki) should be calculated using the standard Cheng-Prusoff equation:

Notes

• Ki carries the same prefix as the IC50 from which it is derived.
• For competitive binding mechanisms, a Ki is required to be reported for radioligand binding assays, which produce IC50’s by 3 or 4-parameter curve fitting methods. It is recommended that IC50’s not be used as the only decision level data for radioligand binding methods.
• For uncompetitive or complex (ill-defined) binding mechanisms, an IC50 is preferred, since one of the main assumptions for the use of the Cheng-Prusoff equation is based on a competitive, bimolecular interaction.

Optional Result Types for Data Publication

The following table includes possible result types that can be used when calculating and publishing data.