HTRF® Assay Miniaturization

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Photo courtesy of Corning

HTRF® reagents are widely used in high throughput screening due to protocol simplicity and ability to be automated. Fluorescence intensity is concentration dependent rather than quantity dependent (such as radioactivity and luminescence) thereby allowing true reduction of reagent usage - simply reduce the volumes of each component. HTRF® assays to be miniaturized while maintaining accuracy and reproducibility. These characteristics combined with lower compound and reagent usage enable more cost effective screens.

Two general rules should be followed to achieve successful assay miniaturization:

  1. Assay component concentrations should remain constant: volumes of all reaction components should be reduced proportionally.

The fluorescent signal is directly dependent on fluorophore concentration. Simply decrease the volumes of each reagent component used per assay. The example below illustrates the miniaturization from 200 to 20µL.

  1. The geometric characteristics of the optical reader head in combination with the type of plate should be considered when deciding final assay volumes.

As shown in the example below, the excitation beam needs to be accurately focused 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 measurement efficiency.

z height focus ai
Recommended volumes
according to plate type
96 regular 200 µl
96 half well 100 µl
384 regular 80 µl
384 low volume 20 µl
1536 well 8 µl


Case Study: IP-One uHTS Miniaturization:
The IP-One HTRF® assay has been successfully miniaturized to 384-well and 1536-well formats (figure 1). The use of low volume 384-well plates is critical for maintaining assay performance of 20 µl final volumes. (also in figure caption) This preserves proper z height focus and ensures superior detection of emissions. Assay volumes can be further reduced to 8 µls (also in figure caption) while sustaining EC50 values (table 1). The concentrations of all assay components were not increased during the miniaturization process. This feature of HTRF® technology is invaluable to reducing screening costs through miniaturization.

  20 µL (384w) 8 µL (1536w)
Standard curve EC50 0.008 nM 0.011 nM
Z' factor at EC90 0.87 0.69
Antagonist D IC50 3.4 µM 3.5 µM

Figure 1 & Table 1: The IP-One HTRF assay was performed using standard protocol conditions in 384- and 1536-well formats. Final assay volume in 384-well low volume plates was 20 µl and 8 µl in 1536-well plates. An antagonist known to inhibit IP1 induction was used at increasing concentrations.
From Tozawa-Takahashi et al.