The scintillation proximity assay (SPA) format has been applied to a variety of HTS assays including antibody-antigen binding, receptor-ligand binding, protein-protein interactions, protein-DNA interactions, and a number of enzyme-based assays . SPA is a nonseparation radioisotopic technique. In many cases, the SPA bead or plate is coated with a ''generic'' reagent that is capable of sequestering the ''target'' of interest near the surface of the bead. When the target binds to its corresponding radiolabeled ligand, the SPA bead is brought within the mean free path distance of the P-particle, and the energy from the radioactive decay is transferred from the ligand to the scintillant in the SPA bead. Upon absorption of this energy, the scintillant emits a photon of light (for a review of SPA technology see Refs. 6 and 65-67).
The main driving forces behind SPA miniaturization are reagent (both biological and chemical) conservation, cost, and reduction in radioactive material usage and disposal. Since this is a ''homogeneous'' assay format it should be amenable for performing SPA assays in miniaturized HTS formats (384-well, 1536-well, or beyond). The scintillants used in the standard SPA beads have been optimized for use in photomultiplier-based detectors. Consequently, the scintillant was designed to emit light with an emission maximum at approximately 450 nm—the region of maximum sensitivity for this type of photomultiplier. CCD cameras, however, are maximally sensitive in the 600-700 nm region and thus required the development of an appropriate scintillating particle. The recent development of the Leadseeker™ SPA beads (Amersham) enables the emitted light to be detected by field imaging CCD cameras.
SPA assays have currently been performed in 96- and 384-well formats  with well volumes ranging from ~200 |L in the 96-well format to ~50 |L in the 384-well format. Miniaturization beyond the ~5 |L/1536-well format is currently limited by detection sensitivity as well as the compatibility with systems for liquid handling.
To evaluate the possibility of miniaturization of SPA in the 1536-well format, a SPA-RT assay has been optimized for substrate, primer/template, enzyme, reaction time, and SPA Leadseeker™ beads used in the assay. Figure 10 shows an enzyme titration curve of the miniaturized SPA-RT assay performed in a 1536-well plate. The total reaction volume was 5 | L, with an additional 3 | L of stop solution containing SPA Leadseeker™ beads added after the required incubation.
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Figure 10 Miniaturization of a SPA assay into a 1536-well plate. Advances in SPA technology have demonstrated the feasibility of miniaturizing a SPA format. In this example, an assay for HIV reverse transcriptase was adapted for SPA. The figure demonstrates a typical enzyme titration curve for HIV RT. As the amount of enzyme in the reaction is increased, the amount of product formed increases linearly up to 2.5 units of enzyme. The figure on top is the image from the assay as detected by the Tundra. The data is presented graphically on the bottom.
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