Assay Development

Time

~3–6 months

Process overview

  1. You submit an application to us.
  2. You develop a primary screen amenable to automation, with a secondary assay in place.
  3. You attempt miniaturization to 384 well format if the current assay is run in 96 well plates.
  4. You demonstrate consistent Z primes greater than 0.5.
  5. You create a written experimental protocol.

What we do

  1. We consult with you in the early stages of assay development.
  2. We collect information from you to determine when assay metrics are suitable for screening.

What you do

  1. You develop the primary assay and secondary functional assay in consultation with us.
  2. You demonstrate the assay robustness and suitability for the high-throughput screening (HTS) format.
  3. You write the primary draft of the automation protocol.

About the Assay Guidance Manual

The free Assay Guidance Manual can help you develop a successful assay. Please read this manual.

About the assay development worksheet

Office spreadsheet icon Assay Development Worksheet.xls

This worksheet is designed for user labs to understand the metrics and plate layouts used in our small molecule high-throughput screening.

It 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.

Step 1: Identify target and activity

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.

Step 2: Identify reference controls

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.

Step 3: Measure DMSO tolerance

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%.

Step 4: Run entire plates

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.

Screening formats

Preferred Required

Fastest to scale and the easiest to move forward into hit-to-lead chemistry:

  • 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

Conditions cannot be changed:

  • 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

Training and reservations

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

Phase 2: Scale and Screen