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Essentials of In Vitro Assay Development

The ability to design, construct, and run specific, sensitive, and robust in vitro assays is crucial in all areas of biomedical research. In the pharmaceutical and biotechnology industries, assays are required to identify and characterize potential new drug molecules.

Regardless of the precise application or the particular molecule to be measured, several fundamental characteristics ought to be taken into consideration whilst developing an in vitro assay. Careful attention should be paid, not simply to the assay itself, but also to the entire workflow that needs to be evolved from sample preparation to evaluation of the information quality introduced via the assay. They are valid irrespective of the kind of assay to be developed (e.g., antibody-based assays consisting of ELISA and electrochemiluminescence, the determination of total protein/protein concentration, enzyme activity assays, cell-based assays or quantitative PCR, or specialized assay including single-molecule activation assay). Similarly, the key factors are valid irrespective of the detection system employed, be it based on absorbance, fluorescence, radioactivity, or even mass spectrometry. Each particular assay has its very own idiosyncrasies; however, by being vigilant towards the factors mentioned, it can assist one to make certain that the assay development is robust, dependable and fit-for-purpose. Often, guidelines and/or widespread discussions about assay improvement using particular technology exists, which includes those for targeted peptide measurements mass spectrometry. Complex cell-based assays that have the purpose of imitating the in vivo cell environment are playing an increasing function in drug discovery and improvement. It is vital to emphasize that the primary concept of in vitro assay development ought to be adhered to, as it allows one to construct robust and dependable assays that generate significant data.

Assay fundamentals

Assay CharacteristicsVital considerations
SpecificityDoes the particular assay detect only the molecule of interest?
SensitivityDoes the assay detect the level of the molecule in the desired specimens?
Dynamic spectrumDoes the levels of the molecule drop within the dynamic spectrum of the assay?
InterferenceDo the constituents of the assay specimen interfere with the assay?
RobustnessIs the assay able to cope with slight changes in assay specimen/apparatus/operator?
ReproducibilityWill the assay exhibit low inter- and intra-assay changeability?
PrecisionIs the assay proficient in precisely determining the total amount/concentration of the molecule?



Having determined precisely what molecule and parameter of that molecule are to be measured, one is required to establish that the assay will measure only what one needs it to measure and not anything else. If the assay is measuring both the preferred molecule and other molecules, then it is viable that steps can be taken to enhance the specificity of the assay.


The assay ought to be sufficiently sensitive such that the level of the molecule falls properly in the dynamic array of the assay. This is a critical factor while determining the assay layout and detection system.

Dynamic spectrum

The range over which the assay readout is proportional to the amount of target molecule in the sample being analyzed. Some samples are required to be diluted or concentrated, to stay in the dynamic range of the assay. Failure to comply with the dynamic range of the analysis will result in incorrect data.


When developing an assay, it is crucial to take into account, aspects that can interfere with the assay readout, leading to inaccurate results such as:

  • In the case of a fluorescent readout, it is crucial to make certain that the specimen to be assayed does not reduce the fluorescent readout.
  • With some protein assays, a common problem that occurs is the interference by specimen constituents, including reducing agents or detergents.
  • In enzymatically-coupled assays, it is crucial to determine that the coupling enzymes do not accidentally measure some element of the specimen other than the desired molecule.

Reproducibility, Robustness, and Precision

To offer reliable, usable information the assay needs to be robust and reproducible. The assay ought to be robust in that it is not unduly affected by modifications in specimen preparation and handling. Equally, it must provide identical results irrespective of the individual operating the assay. The assay should be particularly precise such that the level of variation is as small as possible both on an intra and inter-assay basis.


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