AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery

Abstract

advertising feature an8 | December 2008 | nature methods application notes cell Biology biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads. The binding of the two antibodies to the analyte brings donor and acceptor beads into proximity. Laser irradiation of donor beads at 680 nm generates a flow of singlet oxygen, triggering a cascade of chemical events in nearby acceptor beads, which results in a chemiluminescent emission at 615 nm. In competitive AlphaLISA immunoassays, a biotinylated analyte bound to streptavidin donor beads is used with an antibody conjugated to AlphaLISA acceptor beads. Rapid and simple quantification of analytesAlphaLISA assays are performed following simple 'mix-and-measure' protocols with reduced hands-on and total assay times compared to ELISAs (Fig. 2). Homogeneous AlphaLISA assays eliminate the need for multiple washes to separate bound from unbound assay components. Miniaturization and automationMiniaturization is a key consideration for reducing screening cost and increasing throughput during the drug-discovery process.AlphaLISA assays are truly miniaturizable and automatable, with ELISA is the most widely used detection platform for the quantification of analytes in biological samples. Because they require multiple washes, ELISAs are difficult to adapt to high throughput and automation. Their relatively narrow dynamic range often requires testing more than one sample dilution. There is clearly a need for simple and more robust alternatives for the quantification of biomarkers in a high-throughput screening format. The new AlphaLISA platform has been specifically designed for that purpose for both the research and drug-discovery fields.The AlphaLISA bead-based technology relies on PerkinElmer's exclusive amplified luminescent proximity homogeneous assay (AlphaScreen ® ) and uses a luminescent oxygen-channeling chemistry 1 . AlphaLISA protocols can be set up as sandwich or competition immunoassays. In a sandwich assay (Fig. 1), an analyte is captured by a AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery PerkinElmer's bead-based AlphaLISA® immunoassays are designed for the detection of analytes in biological samples. These chemiluminescent, no-wash assays are ideally suited for miniaturization and automation. They exhibit remarkable sensitivity, wide dynamic range and robust performance that compares advantageously with conventional enzyme-linked immunosorbent assay (ELISA).

Can the MTT Assay Be Optimized to Assess Cell Proliferation for Use in Cytokine Measurement?

“…Optimizing all assay components is important since stimulated lymphocytes can produce a myriad of diverse cytokines when activated and the type of cytokine markedly depends on the nature of the stimulating reagent as well as assay study conditions. 20 The authors evaluated a combination of four different mitogens, alone and in combination, over a range of concentrations with four incubation periods (24,48,72,96 hours) in cultured PBMCs. Upon completion of the studies and quantifying the cell stimulation index and levels of TNFα, it was concluded that the optimal conditions for induced cell proliferation were a medium supplemented with a mixture of PHA:Con A (at a concentration of 5 µg/ mL each) for an incubation period of 72 hours.…”

“…The AlphaLISA ® (Amplified Luminiscent Proximity Homogenous) immunoassay (PerkinElmer, Waltham, MA) is a bead-based assay that was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). 23,24 In this chemiluminescent assay, singlet oxygen molecules generated by high energy laser irradiation (at 680 nm) of donor beads excite acceptor beads. This results in a chemical cascade that yields an emission signal at 615 nm.…”

“…25 AlphaLISA® assays use streptavidin-coated donor beads that capture the biotinylated anti-TNFα antibody and acceptor beads conjugated with a TNFα specific antibody. 24 All are coated in a layer of hydrogel that provides the necessary functional groups for bio-conjugation if the beads are within 200 nm from one another due to the capture of the analyte by the mAb's. 24 The change in light emission is proportional to the concentration of TNFα present.…”

“…24 All are coated in a layer of hydrogel that provides the necessary functional groups for bio-conjugation if the beads are within 200 nm from one another due to the capture of the analyte by the mAb's. 24 The change in light emission is proportional to the concentration of TNFα present. Thus, the use of HTRF and AlphaLISA ® assays regarding the mitogen stimulation of PBMCs may provide greater resolution of stimulation indices when used in conjunction with the MTT assay for cellular quantitation of cytokines such as TNFα.…”

Immunodiagnostics: Major Advances and Future Insights

“…The development of highly sensitive enzyme substrates have led to ultrasensitive ELISAs and chemiluminescent immunoassays [9], while the use of superior fluorescent dyes improved the fluorescent immunoassays. Additionally, the use of infra-red dyes, quantum dots, gold nanoparticles, beads, and other nanomaterial/nanocomposites enabled the development of new ELISA formats, such as the naked-eye ELISA that was demonstrated using gold nanoparticles [10].The development of bead-based AlphaLISA by Perkin Elmer [11] is another major development in immunodiagnostics as it has critically reduced the immunoassay duration and complexity. It employs a streptavidin-coated Alpha donor bead and anti-analyte-conjugated AlphaLISA acceptor bead, which come into close proximity to generate the signal in the presence of analyte.…”