Pharmacokinetic (PK) and Pharmacodynamic (PD) studies play a critical role in the drug discovery and development process. The studies typically require characterization of drug and biomarker concentrations from large numbers of samples with high sensitivity and broad analytical range. The AlphaLISA® technology (PerkinElmer, Inc., Waltham, MA) is a homogeneous no-wash alternative to conventional solid-phase ELISA assays that can be applied to biomarker detection in plasma and serum samples.
In the AlphaLISA assay, a biotinylated antibody and an antianalyte-conjugated Acceptor bead are used to capture the target analyte. The biotinylated antibody associates with a streptavidin-coated Donor bead. When the analyte is present in the sample, the Donor and Acceptor beads are brought together. Upon excitation, a photosensitizer inside the Donor bead converts ambient oxygen to an excited singlet state. Singlet oxygen diffuses up to 200 nm to produce a chemiluminescent reaction in the Acceptor bead, leading to light emission. The amount of light is proportional to the amount of analyte present in your sample, and the assay can be quantified by running against a standard curve.
The AlphaLISA workflow has been demonstrated to provide lower limits of detection, improved assay precision and broader dynamic range compared with conventional ELISA while allowing for a 5-fold reduction in sample volume for measurement of insulin in plasma samples. The technology has been applied in an automated process supporting high- throughput bioanalysis with a capacity in excess of 10,000 data points in 6 hours.
Automation of the AlphaLISA reagent addition steps utilizing the Tempest® microfluidic dispensing instrument allows for a 6-fold reduction in reagent dead volume from 30-65 ml down to 5-10 ml compared with a solenoid valve-based dispensing instrument while maintaining assay performance in the automated workflow.