Requirements

Drug target proposals should meet the selection criteria and the screening requirements listed below.

Selection criteria

The Selection Committee assesses proposals against three main criteria:

  • Is the target or approach novel, or is there a clear need for hits?
  • Are the screening assays and supporting data of the highest scientific quality?
  • Is the assay compatible with ultra-high throughput screening (uHTS)?

Screening requirements

In addition, the proposed screening assay should meet the requirements listed in the table below and the owner should have hands-on experience with the assay.  If the assay does not fully meet the requirements, please indicate in the application form what in your opinion is needed in terms of resources and assistance to bring the assay up to standard. If you have developed a validated 96-well assay but do not have access to 384-well microtiter compatible plate readers or need advice on assay development in general, please contact the European Screening Centre. We are happy to advise on miniaturisation to 384-well format and discuss arranging a site visit for you to upgrade your assay to meet the European Lead Factory standards.

Please note that:

  • Assays in ELISA format are not eligible for submission, unless the assays can be converted to a homogenous format.
  • In case one or more assay reagents are carcinogenic or mutagenic, please search for alternatives in line with the European legal framework.
  • Kinetic measurements on the HTS system have limitations; alternatives are recommended.
  1. Assay requirements for High Throughput Screening
  2. Assay requirements for High Content Screening
  3. Calculating your Z' alias Z prime alias Z-factor

Assay requirements for High Throughput Screening 

 

Characteristic

Requirement

Assay format

Demonstrated in 384-well format, 
scalable to 1536-well format

Homogenous assay

No wash steps

Characterised reference

Available from partner or commercially available

Read out technology

Compatible with mix-and-measure and homogenous formats, e.g.:

  • Absorbance
  • Luminescence
  • Fluorescence intensity
  • Fluorescence polarization
  • Fluorescence resonance energy transfer (FRET)
  • Time resolved Fluorescence (TRF)
  • Homogeneous Time Resolved Fluorescence (HTREF /TR-FRET)

Or alternatives that give highly specific readouts (e.g. AlphaScreen technology, fluorescence lifetime, fragment complementation or FLIPR for calcium readout)

Signal / Background (S/B)
in 384-well plate format
Max assay end volume 30 µl

Sample end concentration 10-5 M

Ideally > 3 or higher

Z' (Z prime)

in 384-well plate format

Max assay end volume 30 µl

Sample end concentration 10-5 M

> 0.6

DMSO tolerance

Minimal tolerance 1.0 % DMSO

Stability of each reagent Stable for at least 8 hours
For proteins: proven freeze-thaw cycle stability
Cell lines

Certified mycoplasma free 
Stable cell lines available for transfer

Certified mycoplasma free 
Stable cell lines available for transfer

CV <10% across plate filled with reference compounds

Protein

Recombinant protein at least 80 % pure

Provided by you, or feasible to produce on milligram scale (construct and protocol available for transfer)

Incubation times

Up to 4 hours

Readout stability For at least 1 hour
Experience Hands on experience in running the assay
Experimental data

Experimental data available  

 

Assay requirements for High Content Screening

 

Characteristic Requirement

Assay Format

Demonstrated in 96-well format, preferably available in 384-well format
Controls
  • Phenotype(s) of interest can be measured quantitatively in the proposed assay with and without stimulus or inhibition.
  • Control(s) are available from the proposer or commercially available and in quantities to allow scale up.

NB The control(s) does(do) not necessarily need to be a small molecule(s).

Read out technology
  • Fluorescence based imaging (confocal or not)
  • Fluorescence resonance energy transfer (FRET)§
  • 2D or 3D, 2D+t, 3D+t
  • Transmitted light imaging e.g. bright field or phase contrast
  • Flow cytometry

§ possible at certain sites only

Assay Robustness

Plate statistics are available for the assay, such as:

  • Heat map of the assay in a plate format

Provide data on inter-plate variance (technical and biological replicate data).

Signal / Background (S/B)

> 2 preferably

Z’ (Z prime)

Z’ > 0

performed with at least 8 wells of positive controls, and 8 wells negative controls.

% coefficient of variation (%CV)
  • Overall %CV (whole or half plate of controls) <10%
  • Column/row %CV data ideally <10% (depending on S/B
DMSO tolerance Minimal tolerance 0.5 % DMSO
Description of phenotypic analysis routine
  • Image processing, analysis and quantification workflows are available (including software used).
  • In case of Flow Cytometry assay, gating and measures are available.
Cell lines
  • All cells are certified mycoplasma free
  • Stable cell lines are available for transfer
  • Demonstrate enough source material is available for screening around 50,000 compounds in 96-well or 384-well format.
  • For iPS-derived cells – special requirements are established (bankable progenitors, differentiation timelines etc)
  • For Co-culture models: special requirements are established.
  • Other unusual growth conditions or technical requirements eg specific plasticware are known.
Other info related to reagents and readouts
  • Details of fluorophores used in staining and or fluorescent proteins used in the assay or in cell lines are known and can be detailed.
  • Stability of assay/labelling reagents (Abs or other dyes) are known.
  • There is a sufficient supply of antibodies and other key reagents.
  • Anticipated treatment time is known.
Readout stability
  • Fixed end-point assay preferred
  • With staining/signal stable for > 1 week at 4 deg C or >1 day at RT.
  • Live cell assay: depends on the readout/stimulus/ timecourse.
Experience Current hands on experience in running the assay
Experimental data Experimental data available

 

Calculating your Z' alias Z prime alias Z-factor

Please note that Z prime should not be confused with Z-score or Z-value!

The Z-factor is an attempt to quantify the suitability of a particular assay for use in a full-scale, high-throughput screen.
The Z-factor is defined by four parameters: the means (µ) and standard deviations (σ) of both the positive (p) and negative (n) controls.

Z' alias Z prime alias Z-factor

not to be confused with Z-score or Z-value!

The Z-factor is an attempt to quantify the suitability of a particular assay for use in a full-scale, high-throughput screen.

The Z-factor is defined in terms of four parameters: the means and standard deviations of both the positive (p) and negative (n) controls (µp, σp and µn, σn).