KeratinoSens™ Assay

The KeratinoSens^TM assay is a cell-based reporter gene assay. It identifies skin sensitizers by assaying the induction of luciferase under the control of the antioxidant response element (ARE) present in the promoter of the human AKR1C2 gene that is known to be up-regulated by sensitizers. The luciferase signal reflects the activation of endogenous Nrf2 dependent genes by the sensitizers. Using well-established light producing luciferase substrates, this assay allows quantitative measurement of luciferase gene induction, which acts as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Creative BioMart provides transferable, reproducible and predictive KeratinoSens^TM assay service to assist the researchers to address the secondary key event of skin sensitization of Adverse Outcome Pathway (AOP).

A skin sensitizer is a substance that leads to an allergic response following skin contact. This can manifest itself as allergic contact dermatitis and is an important field of research for Creative BioMart. KeratinoSens^TM is the first biological in vitro assay for skin sensitization testing validated by EURL ECVAM. Our KeratinoSens^TM service applies for personal care and cosmetics, chemicals, household productions, and pharmaceutical reagents.

KeratinoSens™ Assay Protocol

1. Cells grown in 96-well plates for 24 h

2.Chemicals dissolved in DMSO (solvent)

4. Chemicals added to cells at 12 different concentrations

5. Incubation for 48 h

6. Determination of cell viability

7. Determination of luciferase activity Lysis of cells Addition of luciferin substrate Measurement of light output

8. Considered positive with a >1.5-fold statistically significant luciferase induction

Data delivery:

• Dose response curves for cell viability and luciferase reporter gene expression.
• MEC (minimum effective concentration), AC50, IC30 and maximum response (%) for cell viability.
• EC1.5, AC50 and maximum response (Imax) for luciferase gene expression as well as sensitizing potential classification.

Creative BioMart has been looking to offer a more complete assay and screening service to the researchers and believe our cooperation with them would ultimately expand footprint from their core pharmaceutical and agrochemical markets.

Reference:

1. A. Natsch. The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers--functional relevance and a hypothesis on innate reactions to skin sensitizers. Toxicol. Sci.2010 Feb;113(2):284-92. doi: 10.1093