There are different types of assays used in medical laboratories. These include a variety of tests to determine the presence and concentration of certain substances in a sample.
The assay process aims to present the target in a discernible/measurable form to the discrimination or identification/detection system e.g. by filtration/washing or selective binding etc. The signal is then detected e.g. by light or electron/chemical detection systems.
Here we will discuss the different types of assay used in medicine:
Ion channels and GPCRs with a Ca2+ permeable membrane potential can be measured using a calcium sensitive dye in a FLIPR high throughput imaging plate reader. This technology is a powerful tool for the rapid screening of GPCR and ion channel targets in drug discovery.
The FLIPR calcium assay is an easy-to-use, homogenous, and robust solution to measure changes in intracellular Ca2+ during high-throughput screening experiments. It uses a new fluorophore that is less susceptible to probenecid and provides a larger signal-to-noise window to improve assay sensitivity and throughput.
The Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm is a novel proposal from the FDA, HESI and SPS to replace current clinical ICH-E14 guidelines with a more complete system for screening drug proarrhythmic risks. It integrates automated electrophysiology screening of drugs on multiple cardiac ion channels with in silico predictions and translational assays using beating human induced pluripotent stem cell (hiPSC) cardiomyocytes.
The CiPA protocol uses voltage sensitive dyes to measure compound effects on voltage, tRise and APD 10-90 in spontaneously beating hiPSC cardiomyocytes. A risk score is generated based on these results and then verified by the translational assays.
Calcium assays use fluorescent indicators to measure changes in intracellular calcium levels. Fluorescent calcium-sensitive indicators are compatible with a variety of detection systems including optical microscopy, plate readers and flow cytometers. These assays are widely used in high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited G protein-coupled receptor signalling.
The fluorescence intensity of the indicator increases when calcium levels increase, and can be directly correlated to the amount of calcium released from the cell’s intracellular stores.
The hERG channel, also known as HERG, defines cardiac repolarization and is a substrate for QT prolongation. Virtually all drugs that delay repolarization or cause TdP block the outward current of hERG channels (Sanguinetti et al, 1995). Thus hERG assays have become an established surrogate marker for proarrhythmic potential.
Several indirect methods of detecting hERG-drug interaction have been developed, such as measuring reduced levels of radiolabeled Rb+ efflux (reflecting diminished outward K+ current) in cryopreserved hERG-transfected cells or using voltage-sensitive dyes. These approaches, however, require less well-characterized cell lines and offer lower throughput than assays based on direct measurements of hERG current.