Catalog of ex vivo whole blood stimulation conditions to generate species-specific cytokine controls

Abstract

Abstract Commercially available reagents for the detection of cytokines in pre-clinical species often rely on cross-reactivity from other species. Species-specific cytokine control materials are ideal for use in determining the suitability of the cross-reactive reagents within the intended pre-clinical species. To generate species specific cytokine positive control materials in a non-invasive manner, a systematic catalog of ex vivo whole blood mitogen stimulation and collection conditions was developed. Since naïve matrix (plasma or serum) is unlikely to have cytokines detectable above the lower limit of quantitation (LLOQ), ex vivo stimulation of whole blood using a variety of mitogens was performed in order to produce measureable cytokine release. Plasma harvested at specific time points from these stimulations was then spiked into naïve (species matched) plasma or serum to achieve analyte concentrations within the analytical measurement range of the assay. This ‘spike-in’ sample serves as a model of the intended test system, and allows for evaluation of the ability to detect species-specific cytokines within both plasma and serum matrices. A range of detection was determined for a number of analytes using a range of concentrations of spike-in using commercially available multiplex assays. By producing this catalog of stimulation conditions including multiple mitogens and durations of challenge for optimized collection per analyte, we are able to quickly generate positive control materials for a wide range of analytes across multiple species and thereby have simplified and streamlined the approach to generating these much-needed materials in the preclinical testing laboratory.