Phone: +385 1 8886300

Email: fidelta@glpg.com

Biochemical Screening

In vitro Screening for Improved Discovery and Optimization of Hits & Leads

More than decade of experience and industry track record in biochemical screening and compound profiling. Automatisation and miniaturisation possibilities (utilization of TTP labtech Mosquito nanoliter pipetting platform allows easier setup of the assays - flexible and applicable for low-DMSO-level requirements).
Transfer of as low as 50 nL of DMSO stock solution into a dry plate.
10 µL assay volume in 384-well plate.

Assay development, optimization and validation

Over 20 years of experience in various in vitro compound profiling assays, against vast number of relevant biochemical therapeutic targets in different types of assays:
  • Homogeneous assays (in solution) require the addition of all reaction reagents in one well without washing or removal of reagents before reading the plate (‘one-pot assay’).
  • Heterogeneous assays (on a solid phase) involve multiple steps including plate-washing, centrifugation, and aspiration of one reagent and dispensing of another, usually involving more than one incubation step in the process before reading the plate.

Readout types and screening technologies

The several screening technologies available at Fidelta, assay robustness and performance, together with stringent QC criteria parameters, deliver high-quality research data.
  • Absorbance (colorimetry)
  • Radioactivity
  • Luminescence
  • AlphaScreen (AlphaLISA)
  • Fluorescence
  • FLINT (Fluorescence Intensity)
  • TRF (Time-Resolved Fluorescence)
  • FP (Fluorescence Polarization/Anisotrophy)
  • FRET (Resonance energy transfer) – donor/acceptor
  • Cytometry - when the size metrics of cells are assessed by an image processor

 

Assay robustness and performance

  • Two series of 16 replicates are analyzed: in the absence (total signal) or presence (minimal signal) of Kinase-1 inhibitor, or in the absence of kinase.
  • Target values: %CV of <5 for the total signal, for an S:B ratio from 2 up to >20. These values, combined with a calculated Z'-factor of >0.7, clearly demonstrate the robustness of the optimized assay.

Assay results and next steps

Initial screening aimed at hit generation frequently starts with biochemical binding assays and is followed by functional assays.
 

Data calculation and QC parameters

Calculation of IC50 data, curves and QC analysis are done using Excel tools and GraphPadPrism software, v. 5.03. Briefly, individual dose-response curves are generated by plotting the logarithm of the tested compounds concentration (X) vs. corresponding percent inhibition values (Y) using least squares (ordinary) fit. Best fit IC50 values are calculated using Log(inhibitor) vs. normalized response - Variable slope equation, where Y=100/(1+10^((LogIC50-X)*HillSlope)).

QC criteria parameters (Z’, S:B, R2, HillSlope) are checked for every IC50 curve.
 
 
© 2015 All Rights Reserved - Fidelta Ltd.