Application of CE-C4D for beta-secretase activity assay
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| Year of publication | 2017 |
| Type | Conference abstract |
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| Description | Alzheimer’s disease is the most common form of dementia afflicting more than 35 million people worldwide. It is estimated that prevalence of Alzheimer’s disease doubles every 20 years, but causal treatment is still unavailable. One of the therapeutic targets is ß-secretase, respective its inhibition, resulting in reduction of production of the amyloid ß-peptides. Inhibition assays of ß-secretase are mainly conducted using high-throughput fluorescence microplate readers and labeled substrates permitting fluorescence resonance energy transfer based experiments (FRET). However, FRET-based assays might possess bias originating from fluorescence quenching by tested compounds. As a result, a novel method based on separation of proteolytic products of unlabeled substrate of ß-secretase was developed. The method employs capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D). The sample is online preconcentrated by means of transient isotachophoresis providing limits of detection around 20 nM. The method’s intermediate precision is 3%. Finally, accuracy of ß-secretase kinetic assays was determined by comparing the results with mass spectrometry based assessment. |
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