The iLite® cell-based solutions are based on the iLite technology, a cleverly engineered cell-based assay system with a dual reporter gene readout. They offer the ease of use and robustness of a Ligand Binding Assay and can be developed for virtually any pharmaceutical target and allows an easy, rapid and accurate test format for measurement and quantification of drug activity and immunogenicity.
Cell based assays are defined as any assay that takes place within a living cell. Because this definition is so general, there are thousands of different cell based assays.
Reporter-gene assays are a specific kind of cell-based assay with an engineered system that incorporates a regulatory elements that control a given gene and that use an easily detectable readout like luciferase.
When developing a new cell-based assays, or keeping existing ones alive - there are many hurdles to overcome. Being able to create an assay with a reproducible readout, a high signal-to-noise ratio is challenging and cumbersome which is necessary to achieve sufficient selectivity (few false-positives) and sensitivity (few false negatives), plus high stability.
The iLite® cell-based solutions are supplied as cryopreserved assays ready cells, that are harvested and frozen at the exact same time an higher reproducibility and overall superior performance compared to cells in culture can be obtain – not the mention the time and cost you can reduce but not having to culture your cells!
Start by thawing your assay-ready cells. All cells were harvested and frozen at the same stage which ensures excellent reproducibility and performance. Save time and cost by not having to culture your cells.
Next, plate your cells and add your sample. The cells contain a secondary reporter gene that can be used as a powerful tool for normalizing results and to compensate for differences in cell numbers. This means that you can be confident that you get a target-specific signal.
Finally, incubate and read your assay. The combination of excellent target specificity and a high signal-to-noise ratio ensures a biologically relevant functional assay.
Explore our range of cell-based Functional Bioassays, ADCC system with Effector and Target cells engineered to work optimally together as well as our Gene Therapy solutions.
When a ligand, e.g. your drug candidate, binds to its receptor on the iLite cell surface, a specific intracellular signaling pathway is activated. This triggers the transcription of a specific reporter gene construct coding for luciferase.
When a substrate is added, the luciferase generates light, and the amount of luciferase and thereby the amount and activity of the ligand can then be measured as light emission using a luminometer.
By combining unique features such as a normalization readout and chimeric transcription factors with a highly flexible product format, the iLite technology can help you make the most of your bioassay.
Briefly, the chosen host cell is transfected with target specific genetic constructs and stable clones are produced that carries both a inducible (Target specific) reporter gene and a Constitutive (Normalization gene) as wells as constructs for expression of surface receptors and for engineered transcription factors.
Cell-based assays is a versatile tool for the whole drug development process and there are many reason for conducting cell based assays.
As many of the biological drugs that exists, or are currently under development, have multiple functional domains that interact with several different molecules in order to function appropriately, different tests and validations of the function, efficiency and safety must be performed before the end product is released.
In a regulatory setting, cell based assays are commonly used for cytotoxicity testing, to determine the biological activity (potency) of drug product and drug substance, to determine the mechanism of action (MOA), early stage proof of principal studies, and in immunogenicity studies to determine if antibodies produced by the patient are neutralizing the drug product.
“Generally FDA considers that bioassays are more reflective (than competitive ligand-binding assays) of the in vivo situation and are recommended” (Guidance for Industry: Assay Development for Immunogenicity Testing of Therapeutic Proteins, issued by FDA, Dec 2009.)
“Ideally, the potency assay will represent the product's mechanism of action (i.e., relevant therapeutic activity or intended biological effect)” (Guidance for Industry: Potency for cellular and gene therapy products, issued by FDA, Jan 2011)
“For most biological products, the most appropriate neutralizing antibody assay is a bioassay which measures the neutralization of the bioactivity” (EMA Guidance on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use, issued by EMA, 2012.)
iLite cells are delivered as Assay Ready Cells, and stored at -80°C. There is no need for cell culturing and continuous maintenance of cells – just thaw and dilute before use in the assay.
Besides significant reductions in the amount of labor required and assay turnaround times, the Assay Ready Cell format also gives superior assay reproducibility in comparison to cells in culture, since all cells are cryopreserved at the same passage number.
A second reporter gene used as an internal control will compensate for differences in cell number – you no longer need to count the number of cells in each well.
In addition, it can be used to compensate for serum matrix effects, other complex matrices or if luciferase is sensitive to the compound you are analyzing.
The sensitivity of iLite cells is enhanced through up-regulation of key components, such as the receptor and certain signaling pathway proteins. The up-regulation of receptors also confers a higher specificity, and this is enhanced through the use of chimeric transcription factors and synthetic reporter gene promoters.
These elements are used as a lock and key to transcription of the reporter gene – only the chimeric transcription factor can bind to the synthetic promoter, and endogenous transcription factors are thereby unable to trigger expression of the reporter gene. In this way, pathway cross-talk can be effectively minimized.
The Assay Ready Format not only reduces the assay turnaround times and amount of labor required, but also reduces assay variability in comparison with cells in culture. Traditionally, cell-based assays are known for their high variability, with %CV often over 25%.
Robustness assays of iLite assays have shown that repeatability and intermediate precision are in the range of 4-11% CV, and sample accuracy between 92-107%. In other words, by using iLite Assay Ready Cells, you are getting the biological relevance of a cell-based assay with the ease of use and precision normally associated with a ligand binding assay.
Our cell-based solutions is a versatile tool for applications extending across the whole drug development process and can be applied to many different drug targets.
As every project is different and every development process has its own hurdles to overcome, our custom cell-based development services enables us to offer versatile assay solutions to pharma, biotech and CRO customers, as well as to our partners within diagnostics, tailored to your specific needs.