A wide variety of applications for drug discovery require sensitive, quantitative assays. For example, studying cell signaling pathways
to develop and understand new drug therapies require assays that are able to distinguish small changes in
transcription, molecular interactions and cellular health. For example, studying transcription can involve characterizing promoters and enhancers of transcription factors, identifying genetic point mutations or deletions, or even cellular stress causing changes in
environmental conditions. Knowing how these factors affect pathway outcomes requires the
ability to monitor small and subtle differences between experimental and control samples.
Luminescence, fluorescence and absorbance are the three most common methods used in these assays. In general,
luminescent assays are more sensitive than fluorescent assays, and fluorescent assays are more sensitive than
absorbance assays. Whatever the assay method, detecting low level or small changes in a target requires a sensitive assay. For example, when studying an enzymatic
reaction, you don't want to have to add a high level of substrate enzyme or catalyst to drive the reaction to a detectable
level because this creates an artificial system that does not reflect physiological
conditions. Instead you want your assay to be sensitive enough to be performed at near physiological levels, thus
generating more biologically relevant data.
Sensitive Assays Need Sensitive Detection Platforms
Sensitive assays, however, also require a sensitive detection platform. Plate readers that are used to detect
luminescence, fluorescence and absorbance signals can vary in sensitivity (Figure 1). Factors that contribute to an
instrument’s sensitivity, or limit of detection, include background noise from the detector and
electronics, the type of detector that is used, and the instrument’s configuration and overall design. Lowering the
instrument’s background improves the limit of detection, which in turn improves the signal-to-noise ratio, providing
more usable data from each experiment. Conversely, a high background can over shadow low‐level signals, reducing the usable data from each experiment. To get a true picture of the biology in the cell, it is more relevant to measure your assay at near physiological conditions if possible (Figure 2).
It is easy to focus on the sensitivity of your assay and forget that an assay’s results are only as sensitive as the detection
level of the instrument. The combination of instrument and assay sensitivity provide the true level of
detection for your experiment. Having an instrument with the sensitivity necessary to achieve the level of detection you
need means you will need to use less sample, less enzyme and less catalyst to perform your experiments. You will also
be able to detect fewer cells, lower levels of transcription and more subtle changes in molecular interactions. Sensitivity comparisons (LOD and LOQ) between commercially available detection instruments indicated GloMax® Discover and Explorer exhibited 1 to 2 logs better sensitivity of the instruments tested (Figure 1). The low instrument background and overall design means that the results detected for each well by the GloMax® instruments give you best chance to measure near physiological levels in luminescent assays.