Amplified Recombinant Cell Bioassay For The Detection Of Dioxin And Related Ah Receptor Ligands
Tech ID: 21033 / UC Case 2009-507-0
The Ah receptor (AhR) is a ligand-dependent transcription factor that mediates the ability of dioxin and related halogenated aromatic hydrocarbons (HaHs) to induce gene expression and to produce toxicity. Cell-based bioassays for dioxin-like chemicals (commonly referred to as CALUX bioassays) respond to dioxins and related chemicals with the induction of luciferase in a time-, dose-, AhR-, and chemical specific manner.
A researcher at the University of California Davis has developed and characterized a new CALUX bioassay for the detection of lower concentrations of dioxins and dioxin-like chemicals within a sample.
There is a need for rapid, inexpensive and accurate methods for the detection and quantification of HAHs, such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) and related chemicals in environmental, biological, food and other matrices. This invention provides an improved CALUX cell-based reporting system meeting this need.
The current invention outperforms currently available cell bioassay systems in that it can detect dioxins and related dioxin-like chemicals at substantially lower concentrations (10-100 fold lower) and a significantly higher assay response than that of other bioanalytical systems.
Non-exclusive licenses are available for UC's property rights in this dioxin-detecting cell bioassay and patent rights in the luciferase reporter gene as it is utilized within the dioxin-detecting cell bioassay.
This cell bioassay system also contains components owned by the Promega Corporation. Licensees can acquire Promega permissions relevant to practicing this invention by executing a contract services agreement directly with Promega. UC can provide interested parties with a draft license agreement as well as a sample of the Promega contract services agreement.
While high-resolution instrumental analysis methods are the gold standard method for detection of dioxin-like chemicals and provide very accurate measurements of HAH presence, these procedures require expensive instrumentation, are time-consuming, and are impractical for large-scale screening analysis. This improved CALUX cell bioassay system offers a 10- fold to 100-fold increase in the minimal detection limits for dioxin and related chemicals and a dramatically enhanced overall response compared to other bioanalytical methods. When coupled to the rapidity and very low cost for this assay, it is optimal for high throughput screening applications. The assay can be used for detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of matrices, including primary matrices of concern such as food, feed, biological (tissue, milk and blood) and environmental samples.
Mouse, rat and human hepatoma cell lines, stably transfected with the AhR-responsive CALUX luciferase reporter plasmids, of this invention have dramatically improved sensitivity and reporter gene responsiveness to TCDD and related AhR agonists.
These cell lines have dramatically improved luciferase activity observed at all inducer concentrations. This increased activity and sensitivity not only allows for more accurate determinations of the induction response at the lower end of the concentration response curve, but it also results in a lower limit of chemical detection with these new cells.
The increased response of these cells has also allowed them to be used in a range of microplate formats (96 to 1536 well), reducing the amount of sample and reagents needed for cell growth and analysis while also reducing analysis cost and increasing sample throughput.
- He, G., D. S. Baston, M. S. Denison and T. Tsutsumi. 2008. Third generation calux luciferase reporter vectors - amplification of dioxin responsive elements dramatically increases calux bioassay sensitivity and responsiveness. Organohalogen Compounds. 70, 000772-000775.
- Denison, Michael S.
dioxin, food safety