HTRF reagents are widely used in high throughput screening, assay development and MTS because of their protocol simplicity and amenability to automation. Fluorescence intensity is concentration-dependent rather than quantity-dependent (as is the case for radioactivity and luminescence). This property allows HTRF assays to be easily miniaturized while maintaining their accuracy and reproducibility. These characteristics, combined with lower compound and reagent requirements, enable more cost-effective screens and assays.
Two general rules should be followed to achieve successful assay miniaturization
1The fluorescent signal is directly dependent on concentration. Reagents should not be diluted. Instead, simply decrease the volumes used per assay. The example given here illustrates a miniaturization from 200 to 20 µL. Reagent concentration may be required for a maximum assay performance of ultra-miniaturized assays (lower than 5 µL). Contact our technical teams for more details.
2As shown hereafter, the excitation beam needs to be focused accurately into the well (case A). In case B, where the same plate format is being used with a smaller volume, unfocused excitation will lead to a poorer collection of emitted light. The use of a plate designed for small assay volumes, such as in case C, will restore the measurement efficacy.
Recommended volumes depending on plate types
|Plate type||Assay volume|
|96w regular||200 µl|
|96w half area||100 µl|
|384w regular||80 µl|
|384w low volume||20 µl|
Miniaturization in action
Miniaturization of the cAMP cell-based assay in 1536w plates (10 μL)
Z' factor, as originally described by Zhang et al. (J Biomol Screen. 1999;4:67–73), is now a well-established statistical criteria used to assess the overall robustness of an assay for screening campaigns. It is particularly useful for evaluating the impact of miniaturization. Because they are homogeneous by nature, HTRF assays show better accuracy than other formats (e.g. bead-based or heterogeneous). They give high Z'-factors in most configurations, even at low signal-to-background. The ratiometric correction and the excellent stability of the chemistry also contribute greatly to this robustness.