Mj

Immobilization of membranes containing receptor using proprietary coating procedure

(96- and 384-well FlashPlate microplates)

Incubation with radioligand and compounds

Bound radioligand detected in Top Count™

Figure 21 Schematic representation of FlashPlate technology. Receptor immobilized to a 96- or 384-well FlashPlate is incubated with radioligand and compounds. Receptor bound radioligand is in close proximity with Scintillant and is detected in a TopCount. (Courtesy of Packard, a Packard Bioscience company.)

tors (e.g., human estrogen receptor) [67] interleukin-1 receptor, and G-protein coupled receptors (e.g., endothelin receptors) [68]; (3) radioimmunoassays (e.g., cyclic AMP, cyclic GMP, prostaglandin E2) [69]; (4) functional assays with live cells (e.g., adenyl cyclase assay) [69]; and (5) molecular biology techniques including sandwich hybridization assay and translation systems [69]. For CAT assay, biotinylated chloramphenicol is coated on streptavidin coated FlashPlate. The reaction is done in the chloramphenicol coated streptavidin FlashPlate™ by adding 14C- or 3H-acetyl CoA in the reaction buffer with the addition of CAT, incubated at 37°C and counted after incubation. The plate is counted again after aspiration of the liquid and washing with buffer. The counts detected in chloram-phenicol are similar either with or without aspiration of the reaction medium, suggesting that the assay can be done without aspiration and washing in a homogeneous mode [66].

3. Comments

FlashPlate technology can be used for a wide variety of applications such as enzyme, receptor binding, functional and immunoassays and in live cells. Com monly used radioisotopes (i.e., 3H, 14C, 35S, 125I, 32P, 33P, and 45Ca) can be used with FlashPlates. With low energy beta emitters such as 3H, 14C, and 35S isotopes, FlashPlates can be used in homogeneous mode. However, with higher energy beta emitters such as 32P and 33P, the unbound radioactivity should be removed by aspiration and rinsing, because these radioisotopes can be detected due to the long distance traveled by the strong beta particles and may interfere in the assay with high background. Availability of generic FlashPlates precoated with commonly used proteins such as streptavidin, protein A, antibody and MBP and nickel chelate in assay ready format is an added advantage. Preparation of a custom biomolecule bound to FlashPlate involves binding the compound and several washings, which is labor intensive, and batch-to-batch variations may occur.

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