T-lymphocyte mediated drug hypersensitivity reactions mainly target the skin and liver1,2. These reactions are dependent on antigen presentation to drug-specific T-lymphocytes which result in their activation, expansion, differentiation and the targeting of tissues and organs by activated T-lymphocytes.
The peripheral blood mononuclear cells (PBMC) proliferation assay is an in vitro method which utilizes PBMC isolated from consenting healthy donors using Ficoll and density gradient centrifugation. This assay is also suitable for evaluating the priming of naïve T-lymphocytes within the PBMC population and for the assessment of immunogenicity and cross reactivity of small chemical molecules, biologics and peptides. In addition, inter-individual variability in immune responses can be explored by using multiple HLA-typed donor PBMC. Furthermore, follow-up studies involving other endpoints, such as drug-induced cytokine release profiles can be performed using the same cryopreserved donor PBMC.
Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.
In vitro high throughput assays utilising primary human immune cells will significantly enhance our capabilities to predict candidate drugs with potentials to cause rare but occasionally fatal hypersensitivity reactions during early stages of preclinical drug development.
|Donors||>6 donors available for multi-donor studies|
|Analysis Platform||PBMC proliferation – fluorescent detection using Alamar blue cell viability reagent
Cellular ATP – CellTiter-Glo® (Promega) with a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek)
|Test Article Concentrations||8 point dose response curve with top concentration based on 100x Cmax or solubility limit
3 replicates per concentration*
|Test Article Requirements||Maximum (dependent upon number of repeat doses) 150 µL of a DMSO* solution to achieve 200x top concentration maintained at 0.5% DMSO or equivalent amount in solid compound|
|Time Points||24-72 hour pre-incubation*|
|Quality Control||Negative control: 0.5% DMSO (vehicle)*
Positive controls: 2 appropriate compounds
|Data Delivery||Minimum effective concentration (MEC) and AC50 value for PBMC proliferation and cellular ATP content|
* other options available on request.
1 Sullivan A et al., (2018). β-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells. J Allergy Clin Immunol 141(1); 235-249
2 Mennicke M et al., (2009). Fulminant liver failure after vancomycin in a sulfasalazine-induced DRESS syndrome: fatal recurrence after liver transplantation. Am J Transplant 9(9); 2197-2202.
3 Gibson A et al., (2018). Genetic and nongenetic factors that may predispose individuals to allergic drug reactions. Curr Opin Allergy Clin Immunol 18(4); 325-332.
4 Pichler WJ and Tilch J, (2004). The lymphocyte transformation test in the diagnosis of drug hypersensitivity. Allergy 59(8); 809-820
5 Nyfeler B and Pichler WJ, (1997). The lymphocyte transformation test for the diagnosis of drug allergy: sensitivity and specificity. Clin Exp Allergy 27(2); 175-181