Section5:Introduction

From Assay Guidance Wiki

There are two typical assay formats used for analysis of receptor-ligand interactions in screening applications, filtration and scintillation proximity assay (SPA). Both formats utilize a radiolabeled ligand and a source of receptor (membranes, soluble/purified). Receptor binding assays using non-radioactive formats (fluorescence polarization, time-resolved fluorescence, etc.) which are continually being investigated for feasibility, would have similar assay development schemes to those presented in this document.

Selection of the detection method to be used (SPA, filtration, non-radioactive) is the first step to receptor binding assay development. In some cases, investigation into more than one format may be required to meet the following desired receptor binding criteria:

• Low nonspecific binding (NSB)
• > 80% specific binding at the Kd concentration of radioligand
• Less than 10% of the added radioligand should be bound (Zone A)
• Steady state obtained and stability of signal maintained
• For competition assays, the radioligand concentration should be at or below the Kd
• No dose response in the absence of added receptor
• Reproducible
• Appropriate signal window (i.e. Z-factor > 0.4, SD window > 2 SD units)

While developing receptor binding assays, some of the experiments may need to be performed in an iterative manner to achieve full optimization. In addition preliminary experiments may be required to assess the system.

In many instances, a multi-variable experimental design can be set up to investigate the impact of several parameters simultaneously, or to determine the optimum level of a factor. It is strongly recommended that full assay optimization be performed in collaboration with an individual trained in experimental design.

Experimental design and assay variability is addressed in detail in other sections of this handbook.

The following pages should be used as a general developmental guide to receptor binding assays using SPA or filtration formats.