The major constituent of senile plaques associated with Alzheimer’s disease is the beta-amyloid peptide, derived from the amyloid precursor protein (APP) by proteolytic cleavage. This cleavage occurs at the beta and gamma cleavage sites. Early biochemical characterization and inhibitor profiling have indicated that both beta and gamma cleaving enzymes (secretases) are probably aspartic proteinases, and are currently the most attractive targets for therapeutic intervention for Alzheimer’s disease.

The QTL Lightspeed beta-secretase assay combines high-sensitivity with high speed for screening potential inhibitors against b-secretase. The assay is based on QTL’s patented “superquenching” technology (see movie).

Assay Description
The QTL Lightspeed protease assay makes use of fluorescence-quenching protease substrates. The peptide sequence is recognized by the enzyme of interest and separates the QTL fluorescent polymer from our proprietary molecular quenchers. Proteolytic cleavage of the peptide releases the quencher from the QTL polymer, resulting in a quantitative signal that increases with enzyme activity or reaction time. Inhibitors for the enzyme activity modulate this signal increase, which can be tuned over a variety of sensitivity ranges as desired by target sensitivity requirements.

Detection Requirements
The QTL Lightspeed protease assay can be used in virtually all commercially-available fluorescence spectrometers and multi-well plate readers. The excitation (blue) and detection (green) wavelengths are compatible with all common fluorimeters. QTL technical personnel can assist with configuring the settings to optimize performance of the QTL Lightspeed assay.

Trade-off possibilities (Time vs Sensitivity vs Compound Requirement)
Because enzyme activity is catalytic, or based on the turnover of multiple chemical reactions with a single catalyst, the increase in fluorescence in the QTL Lightspeed assay occurs more rapidly in the presence of larger concentrations of target enzyme. Because of this effect, a more sensitive assay platform allows the user to choose the optimal parameters for a particular screening assay. For example, if moderate sensitivity is required, the assay can be performed at much higher speed, whereas much higher sensitivity can be achieved at moderate speed. The QTL Lightspeed b-secretase assay performs at least four times faster than competing assays at equivalent sensitivity.