(1345-B) Miniaturization of fluorescent kinetic assay for optimal conditions: maximizing fluorescence while minimizing volume.
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: AssayQuant Technologies’ core business focuses on the PhosphoSens® assay that relies on proprietary sensor peptides. When a sensor peptide is phosphorylated by a specific kinase, chelation of a magnesium ion is enabled, resulting in increased fluorescence of the fluorophore and a continuous activity readout. These kinetic assays are the foundation for AssayQuant’s Potency and Selectivity testing services.
AssayQuant’s current standard assay protocol utilizes a 384-well polystyrene plate with an assay volume of 20 µL. Standardization of the assay plate type and the total reaction volume has enabled AQT to simplify assay development of new sensor peptides, introduce automation for routine workflows, and leverage bulk consumables purchasing. However, in an ever-changing market, additional cost-saving avenues were identified and evaluated to help mitigate supply chain challenges and improve profit margins.
Systematic evaluation of the standard assay was performed in a quantitative PCR (qPCR) reaction plate as an alternative to the standard format. The qPCR plate that was chosen is comprised of two different plate materials: a polycarbonate frame that allows the plate to be utilized within robotic systems, and polypropylene wells that allow for fluorescence detection. Assay volume and concentration ratios between sensor peptide and kinase concentrations were investigated for their potential effects on fluorescence intensity and reading confidence. Using an understanding of the effect of reduction of assay volumes, the assay was compressed to a 1536-well format with a final reaction volume of 5 µL, maintaining assay integrity.
AssayQuant determined viable assay conditions that employ different plate types and assay volumes to ensure that the assays are as efficient as possible in a variety of assay formats (384-, 1536-well), while maintaining adequate fluorescence output and minimizing assay cost.