Luciferase Measurements in High Throughput Screening

Abstract

Luminescence assays are becoming more popular in high throughput screening (HTS) laboratories with the luciferase reporter gene being the most common. As with other assays that are adapted to HTS, improvements have been made to the luciferase assay to make it better suited to the requirements of HTS. For the luciferase reporter gene, these improvements included stabilization of the enzyme, increasing the half-life of the luminescence signal to 5 h, and eliminating separation steps (centrifugation and aliquot transfer) after cell lysis. The improved assay, LucLite, is homogeneous and is measured directly in the cell culture media. In addition to reagent improvements, a temperature-controlled, multidetector microplate counter, TopCount, can quickly and accurately measure luminescence signals.

Homogeneous Proximity Tyrosine Kinase Assays: Scintillation Proximity Assay versus Homogeneous Time-Resolved Fluorescence

“…The resulting HTRF ratio was only marginally affected by the colored samples even at concentrations up to 100 M (data not shown). In fact, HTRF has been used to assay extremely colored samples such as natural products (22) and human sera (23).…”

High‐Throughput Screening

“…These ions possess unique properties which make then especially attractive for bioassays: they are hydrophilic and have long excited-state lifetimes which enable them to be easily distinguished from other sources of fluorescence emission in a homogeneous sample [28]. Using these lanthanide ions, in the mid-1990s homogeneous assays have been developed for application to HTS [38]. By labeling one component of a competitive binding bioassay with one of these ions, read-out methods are being developed for a range of assays based on the time resolved analysis of fluorescence [28].…”

Use of a Phosphotyrosine-Antibody Pair as a General Detection Method in Homogeneous Time-Resolved Fluorescence: Application to Human Immunodeficiency Viral Protease

“…An HTRF assay for HSP-1 protease employed a similar approach except that rather than relying on indirect detection through phosphotyrosine, the substrate was directly labeled with the Eu cryptate (26). However, we decided against this strategy due to the potential for the Eu cryptate (FW Ϸ 1400) to modulate the substrate's suitability, the necessity of incorporating the label after synthesis on resin, and the potential generality of using a labeled antiphosphotyrosine antibody.…”

“…The optimization of the HTRF detection step and role and molecular interactions of the different components are also described. A related approach utilizing a biotinylated, europium-labeled peptide substrate for the herpes simplex protease (HSP-1) has been described previously (26).…”