Luminescence-based assay

Researchers at SRMLSC recently developed a HTS that allowed the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries [34], The luminescent-based assay, which measured the inhibition of SARS CoV-induced cytopathic effects (CPE) in Vero E6 cells, was validated with two different diversity sets of compounds against the SARS CoV. The hit rate for both libraries was approximately 0.01%. 

The use of QSAR descriptors for prediction and understanding of the activity of antimicrobial peptides has previously been limited to comparisons between peptides that differ in only a small number of amino acids. This has been primarily due to the cost and difficulty of producing large numbers of peptides as well as the cost of assaying their activity. However, with the recent advance in high-throughput peptide synthesis technique in combination with rapid assay of activity with the luminescence-based assay, robust amounts of data have begun to be available (35). 

High throughput flash luminescence readers such as the FDS6000 and 7000, as well as the Lumax Flash HT, enable functional GPCR and calcium channel testing. A sensitive photon-counting CCD camera enables aequorin and luciferase activity to be measured by flash luminescence. Thus, these advances allow fluorescence-based assays to be replaced by luminescence-based assays,... 

An HTS campaign of 66,000 compounds using a luminescence-based assay was performed under the MLSCN... 

Numerous methods are available to identify and quantify the extent of mitochondrial dysfunction in hPT cells, such as measurements of ATP concentrations by colorimetric or luminescence-based assays, rates of substrate-dependent... 

Well assay plates SoUd white plates are recommended for luminescence-based assays whereas black plates are recommended for fluorescence assays. 

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

Traditionally, lanthanide-based assays have used ions which emit light in the visible region of the spectrum. Europium has been favored over other alternatives since it has a long luminescence... 

Kuemer J.M., Wolfbeis O.S., Klimant I., Homogeneous Luminescence Decay Time-Based Assay Using Energy Transfer from Nanospheres, Anal. Chem. 2002 74 2151— 2156. 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

In order to avoid the described problems, antibody-based assays have been developed. As mentioned above, usually a primary antibody recognizes the modification, which in turn is then quantitated. One of these approaches is based on the technology of AlphaScreen (Amplified luminescent Proximity Homogeneous Assay) [51, 52], also known as luminescent oxygen channeling immunoassay (LOCI) [53], a method that can be used to study protein-protein interactions in general. In this case the enzymatic transfer of acetyl groups to a histone peptide is determined. 

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