Biomedical Engineering Reference
In-Depth Information
Interestingly, the concentration of the limiting component in the assay also affects
the accuracy of the HTS results, since it sets a lower boundary for the EC
50
values
observed in a dose-response assay [9]. Therefore, achieving the lowest possible
concentration of the limiting component is beneficial for SAR and MOA studies. The
supporting components of the assay could frequently help decrease the LOD for the
limiting component, allowing for utilization of a lower enzyme concentration and
thus decreasing the lower boundary of the EC
50
values.
12.3 COMMON ASSAY METHODS AND TECHNIQUES
HTS assays link cellular phenomena or macromolecular functions with quantifiable
physical parameters of biological systems (as shown in Figure 12.1). Diverse biolog-
ical processes such as cell death, translational activity of genes, enzyme activity, and
protein-protein interactions can be characterized in HTS assays reliably and quanti-
tatively. In their turn, these biological processes could be coupled with a variety of
detection signals, resulting in a wide diversity of HTS assays. The major groups of
HTS assays, together with their detection techniques and biological phenomena, are
discussed in this section.
12.3.1 HTS Detection Approaches
HTS assays are commonly grouped according to their detection approaches. Detec-
tion based on spectrophotometric properties is by far the most common in screen-
ing, in major part due to technical and technological simplicity of instrumental
implementation. A variety of monochromator-, optical filter-, or diode array-based
spectrophotometers provide a basis for the flourishing of spectroscopic HTS assays.
Absorbance, fluorescence, and luminescence phenomena are utilized in building HTS
assays. Colorimetric assays, based on the absorbance of light, dominated the field of
spectrophotometric detection in the past; however, fluorescence and especially lumi-
nescence approaches, due to their unparalleled sensitivity, have gained significantly
in popularity over the past decade.
Fluorescence approaches utilize reemission of the light energy absorbed and stored
temporarily by a fluorophore. Three major HTS fluorescence approaches are fluo-
rescence intensity (FI), fluorescence polarization (FP), and fluorescence (Forster)
resonance energy transfer (FRET). While FI makes use of a routine excitation-
emission phenomenon, FP assays utilize depolarization of light through reemission
to gauge the rate of molecular rotation of fluorophore molecules, providing a basis
for binding studies. Given that a significant mass change is required to generate a
reliable FP assay, these assays usually employ low-molecular-weight ligands bind-
ing to large macromolecules. The third type of fluorescence approach, FRET, relies
on nonradiative energy transfer between fluorescence donor and acceptor moieties
to study biomolecular interactions. This energy transfer requires dipole-dipole cou-
pling; importantly, the efficiency of energy transfer is inversely proportional to the
