Small Molecule Discovery Center
SMDC Home / Screening Core / HTS Workflow / Assay Development
Assay Development

Guidelines for assay development can be found at the NIH National Chemical Genomics Center. Please review their Assay Guidance Manual for detailed information.

The SMDC Assay Development worksheet can be downloaded from the documents section. This document describes the calculations used to measure the quality and consistency of high throughput small molecule screening assays (coefficient of variation, Z prime, Z factor). It also shows sample data from a typical 384 well plate taken from an enzyme inhibition screen. The activity of each sample compound is expressed as a percent inhibition value relative to positive and negative reference controls located in the outer columns of the plate. A scatter plot of the plate shows percent inhibition values of each of the 320 compounds tested. The graph displays outlier compounds, or 'hits', which are defined in this instance as inhibition values greater than or equal to 3 standard deviations above the inhibition mean of the set of 320 compounds tested. Finally, the spreadsheet shows our standard quad mapping procedure and our canonical plate layout for positive and negative reference controls.

The first step in assay development is to identify your target and its activity, and then decide if the goal of the small molecule screen is to inhibit or activate your target. If you possess an in vitro end-point enzymatic assay with a robust signal you would like to inhibit, this is usually the easiest type of assay to scale with appropriate positive and negative reference controls. Other types of assays, such as cell-based phenotypic assays, are more challenging to set up, scale and interpret since viable organisms must be uniformly deposited and maintained in microtitre plates and off target compound effects such as toxicity can confound the results. In addition, certain types of homogenous binding assays can be problematic because non-specific binding cannot be quantified and compound interference effects (such as autofluorescence) can swamp the specific signal.

The next step during the assay development phase is to identify suitable positive and negative reference controls to use in your assay. Traditionally, many biologists associate the term positive control with a maximum assay signal and the term negative control with a minimum signal. For purposes of consistency in screening, we define the positive control as being equivalent to what we would consider a "hit". For example, in a typical enzyme inhibition assay, we define the positive control as the enzyme + substrate + inhibitor, while the negative control is defined as the enzyme + substrate in the absence of inhibitor. For a typical enzyme inhibition screen, the ideal positive control would be a potent small molecule that inhibits the enzyme in the presence of substrate in a reliable fashion, while the negative control is usually the enzyme in the presence of substrate and DMSO. Since our compound libraries are dissolved in DMSO, the reference controls should contain the same amount of DMSO solvent as the sample compounds being tested.

Because all samples and reference controls contain DMSO, it's very important to measure the DMSO tolerance of your assay. Whole cell assays normally tolerate no more than 1% DMSO, while biochemical assays can withstand higher DMSO concentrations. The recommended final working concentration of DMSO for biochemical assays is between 0.5%-5%.

One of the last major steps in assay development is to run entire plates each of the positive and negative controls using our bulk dispensers to aliquot reagents into plates. This step is necessary to evaluate the reproducibility and precision of your assay. Coefficients of variation can be calculated, in addition, you will easily spot any positional plate effects and systematic pipetting errors occurring in your methodology.

Listed below are our preferred formats for screening, meaning that these are the types of screens that are the fastest to scale and the easiest to move forward into Hit to Lead Chemistry.

Preferred Formats for Screening

  • 384 well plates
  • 1 µM, 10 µM, or 30 µM final compound concentration
  • Enzyme or other well characterized target
  • End point or equilibrium assay
  • Homogenous assay
  • Scaling is feasible to a minimum of 40 plates per day 3 times a week during normal work hours (Mon-Fri 8-6). Scaled assays should maintain consistent Z prime values greater than 0.5
  • Appropriate secondary assay in place

Listed below are our required formats for screening, meaning that the listed conditions cannot be changed.

Required Formats for Screening

  • Final DMSO concentration between 0.5-5%
  • Compounds in primary screens are run in singlets only
  • Positive and negative reference controls are run in replicates of 8-16 in wells located on the outside columns of the microtitre plates
  • Each individual plate contains positive and negative reference controls
  • Assay data is in text (comma or tab delimited) or Excel file formats

UC users can be trained by SMDC personnel in the use of HTS equipment. This equipment can be reserved using our online calendar. Please contact for more information.

Please refer to the Assay Development section of the HTS Screening Workflow Outline found in the documents section for a summary of the overall process and the distribution of responsibilities between the SMDC and user labs.