Assay Development

Researchers develop assays to conduct primary and secondary hit screening. Given that the data provided by the assay will determine whether a compound or molecule is deemed a “hit”, proper assay development is absolutely essential.

Several factors should be considered when developing an assay for hit screening:

What is a Hit?

Drug discovery and development largely center on finding agents that will modulate targets in order to evoke the desired physiological effect. Are the researchers screening for compounds that interact with the target? Is there a response/potency magnitude threshold that must be met? Is there a specific mechanism of action that must be triggered?1 Should the assay be qualitative, semi-quantitative or fully quantitative?

How Have the Candidates Been Prepared?

Storage and sample preparation conditions will depend on the individual properties of the candidate agents. For example, chemical compounds are often stored in organic solvents such as ethanol or dimethyl sulfoxide (DMSO). When designing a screening assay, researchers need to consider whether elements in the sample preparations, such as these solvents, can exert detectable effects on the target.

How Robust is the Assay?

Several parameters help determine the robustness of an assay.

First, how pharmacologically relevant is the interaction the assay detects? Are the positive controls known to interact with the target and trigger pathogenic mechanisms?

Second, how reproducible are the assay results? For drug discovery, assay results must be reproducible across replicates (in confirmatory runs), experimental runs, across days, and throughout the duration of the program. 

Third, what are the required levels of sensitivity and specificity? Different assay techniques and technologies have varying precision and accuracy limits. Will this assay generate many false positive and negative results?

Finally, how will researchers collect, process and analyze the data? How much statistical confidence can be placed in any trends present in the data?

What are the Throughput Requirements?

The potential number of candidate agents can be extremely high, making high-throughput assay techniques valuable for hit screening.

When determining the necessary throughput capacity of an assay, researchers must consider scientific considerations—such as whether the screen is exploratory or confirmatory, and whether sufficient data will be generated for statistical rigor—and logistical concerns, such as the availability of necessary equipment, and the required labor, time and reagent costs. Moreover, assay developers need to ascertain the flexibility of an assay; is scaling up or down possible? Can the assay protocol be automated now or in the future?

Incorporating Automation into Assay Development

The ability to automate all or part of an assay protocol can offer scientific and logistical benefits.

For example, automating the liquid handling components of an assay using a Biomek Workstation not only increases assay speed, precision and consistency, thereby increasing data quality, it also lessens the ergonomic and time demands of the laboratory staff, thus improving productivity.

The ability or potential to introduce automation into any workflow greatly increases the flexibility and capacity available to assay designers.


  • Assay development is essential for accurate and trustworthy hit generation
  • Deciding what parameters must be met in order to constitute a “hit” is the first step
  • Assay developers must also consider variables such as sample preparation, pharmacological relevance, statistical rigor and logistics
  • Automating an assay workflow can offer scientific and logistical benefits, resulting in better data and better productivity


  1. Hughes JP et al. Principles of early drug discovery. Br J Pharmacol 2011;162(6): 1239-49.

Featured Video: How Automation Helps to Improve ELISA

Products for Assay Development

Biomek i7  Automated Liquid Handling Workstation

Biomek i7

(Hybrid) Flexibility to meet changing assays and throughputs
Biomek iSeries Automated Liquid Handlers

Biomek Software

Simple creation and editing of protocols for new assays

Biomek Method Launcher

DeckOptix FinalCheck ensures correct deck setup for ever-changing assays

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