Development of a High-Throughput Screening Assay for Inhibitors of Aggrecan Cleavage Using Luminescent Oxygen Channeling (AlphaScreen™)

Abstract

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

“…Since increasing AlphaLISA reagent concentration can increase immunosensing sensitivity, the AlphaLISA reagent concentrations used in this work were greater than recommended values by the supplier. Readers interested in AlphaLISA assay optimization can refer to studies published previously34353637.…”

A microplate assay for the screening of ADAMTS-4 inhibitors

“…However, we easily produced different batches of rabbit affinity-purified anti-ARGS-VIL of consistent affinity for the epitope. Furthermore, it also has the advantage of not being based on a proprietary technology, as is the already described HTS assay (Peppard et al, 2003). In conclusion, we present in this paper an immunoassay well suited for rapid screening of aggrecanase inhibitors in chemical libraries.…”

“…This paper presents a protocol for testing inhibitors of aggrecanase activity. Previous HTS assays (Peppard et al, 2003) used a proprietary technology (AlphaScreen™) requiring the development of beads that end up producing a time-resolved fluorescent signal between antibodies linked to these beads and directed against the chondroitin and keratan sulfate regions of aggrecan. The region between these zones is the target of aggrecanase cleavage of aggrecan, leading to the decrease of the fluorescent signal between the beads, since the cleavage physically separates the zones against which the antibodies were raised.…”

Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library

“…High throughput screening (HTS) would be needed to properly find agents that will inhibit the collagenase activity of degradative snake venoms. Similarly, the search for better matrix metalloprotease assays continues (Peppard et al, 2003a(Peppard et al, , 2003b.…”