Orthogonal Assays

In hit identification process, false positives may be caused by spurious, non reproducible activity or the reproducible interference of compounds on primary screen. The initial activity of the compounds in primary screen may also depend on the form of the test rather than on the biology of interest. To further eliminate false positives and confirm the activity, orthogonal assay is performed following the primary screen. Orthogonal assays are used to distinguish between compounds that generate false positives and those that can genuinely active against the target. These assays are conducted on compounds found active in primary screen, but different reporter or assay format is used to confirm that activity of the hit compound is directed toward the biological target of interest. Thus the selected inactive compounds in orthogonal assays need to be removed from further consideration.

Because a significant number of compounds fluoresce or absorb in the wavelength ranges typical of assay sensors, false positives may also occur because the assay response to a compound can be artificially increased or decreased. Orthogonal assays are also useful to eliminate compounds that interfere with the optical properties of the experimental readings. Biophysical techniques are often used in orthogonal assays to confirm the direct interactions between hit compounds and targets because they are largely insensitive to the optical properties of compounds.

Three potential binding patterns from SPR Figure 1. Three potential binding patterns from SPR. (Hevener, K. E., et al., 2018)

Creative Biolabs provides orthogonal assays for hit validation of drug discovery process thus to deal with data reproducibility crisis which is a big obstacle for drug research and development and may lead to failure and high risk of investment. We can drive forward and speed up your drug discovery process by eliminating the false positives and confirming the activity of multiple hit compounds.

Creative Biolabs offers a wide range of technical platforms for orthogonal assays, including but not limited to the following:

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) is a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. SPR uses polarized light to measure angle change. The change in angel is proportional to the change in refractive index of a metal surface and its vicinity upon the binding of any partners. SPR can monitor the real-time interactions between protein–protein, protein–peptide, protein–DNA/RNA and protein compound. We have established an advanced SPR technology platform with several services for drug discovery.

Thermal Shift Assay (TSA)

Thermal shift assay (TSA), also known as differential scanning calorimetry, refers to quantifying the variation in thermal denaturation temperature of a protein in different surroundings. TSA is another method that can be used to investigate specific binding of a compound to a target protein and it has high-throughput capability. We have established a comprehensive thermal shift assay platform with years of experiences in drug discovery.

Isothermal Titration Calorimetry (ITC)

Isothermal titration calorimetry (ITC) is a physical technique used to determine the heat changes caused by interactions in solutions. Compared with SPR, ITC does not require immobilization and modification of molecules, while SPR requires immobilization of one of the binding partners on a sensor surface. In addition, ITC is not limited by the size of proteins or ligands and not interfered by any potential optical properties of the compounds. Therefore, ITC has become a valuable technique in drug discovery.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is a spectroscopic technique based on the phenomena of nuclear magnetic resonance to observe local magnetic fields around atomic nuclei. NMR spectroscopy can provide specified information about the dynamics, reaction state, structure, and chemical state of molecules. NMR has become a preferred method for identifying fragments that specifically bind to a protein or nucleic acid targets for even weak ligands.

X-Ray Crystallography

X-ray crystallography is a useful technique for determining the atomic and molecular structure of a crystal. The ultimate value of this approach is the visualization of compounds and binding details. This can indicate vectors for the expansion and evolution of hits.

Creative Biolabs provides orthogonal assays to confirm the activity of hit compounds using a different assay or technology according to the primary screen of your drug discovery project. We have a dedicated assay development team that will work closely with you to design customized assays. Our service will meet your specific needs fast at extremely competitive prices. If you need more information, please feel free to contact us at anytime. We look forward to working with you to help your drug research and development project succeed.


  1. Hevener, K. E., et al., 2018. Hit-to-Lead: Hit Validation and Assessment. Methods in Enzymology, 610, pp. 265-309.

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