(Under the assumption of normality, one would expect approximately 5%, or approximately 3, of the values to fall outside 1.96 standard deviations of the mean). overall variance in the ED50 values for the in-house reference standard from 55 impartial estimates performed over the period of one 12 months was 12.3% of the average. Excellent intra-plate and within-day/inter-plate regularity was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate regularity in the parameter estimates corresponding to the lower and upper asymptotes as well as to the slope factor at the mid-point. The ED50 values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data units from formal statistical assessments, and to set up improved assay acceptance criteria based on comparability and regularity in the four parameters of the model. The assay is usually precise, accurate and strong and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability. Keywords: Cell proliferation assay, IL-15, Assay qualification, R statistical language and Environment, Statistical analysis 1. Introduction Several cytokines and/or cytokine derivatives have proven to be successful in the medical center as biotherapeutics. Interlukin-15 (IL-15) is usually a cytokine that plays a significant role in lymphocyte homeostatic and NK cell development. IL-15 is usually a 14C15 kDa member of the four -helix cytokine family with structural similarities to IL-2 (Rosenberg et al., 1994; Bamford et al., 1994). Like IL-2, IL-15 stimulates the proliferation of CD4+ and CD8+ T cells, immunoglobulin M (IgM) or CD40L-treated B cells, as well as the generation and persistence of NK cells (Grabstein et al., 1994; Waldmann et al., 2001; Waldmann and Tagaya, 1999; Fehniger and Caligiuri., 2001a; Fehniger and Caligiuri 2001b; Fehniger et al., 2002; Carson et al., 1997., Waldmann, 2006). Mice deficient in IL-15 exhibit depleted NK, NKT, T, CD8+ and memory phenotype T-cell figures, emphasizing the important role of IL-15 in lymphocyte subset development (Lodolce et al., 2002). IL-15 also plays a critical role in the functional maturation of both macrophages and dendritic cells DCs1 (Ohteki et al., 2001). IL-15 enhances the phagocytic activity of monocytes and macrophages and induces the production of pro-inflammatory factors such as IL-8 and MCP-1 (Badolato et al., 1997). In DCs, IL-15 up-regulates the expression of co-stimulatory molecules and IFN-, enhancing the ability of DCs to activate CD8+ cells and NK cells (Mattei et al., 2001, Jinshui et al., 2003). In addition, reduced numbers of peripheral DCs have been observed in IL-15?/? and Erythropterin IL-15R?/? mice suggesting a role for IL-15 in DC survival (Dubois et al., 2005). IL-15 is an immunoregulatory cytokine that exhibits pro-inflammatory activity by acting on a wide variety of cell types. Some of these effects are direct and include Th1 and Th17 polarization as well as activation of effector cells such as B, NK, mast cells and neutrophils. Other effects are indirect and involve the production of other pro-inflammatory cytokines such as IFN- and IL-17 as well as IL-18. Waldmann (2002) demonstrated that, in contrast to IL-2, IL-15 does not contribute to AICD or the maintenance of Treg cells. In light of these functional differences, IL-15 may be superior to IL-2 in the therapy of cancer and as an agent for use in the treatment of patients with AIDS receiving HAART therapy. A number of studies in murine models have suggested that IL-15 may prove to Rabbit Polyclonal to DECR2 be of value in the therapy of neoplasia (Fehniger et al., 2001a; Munger et al., 1995; Kobayashi et al., 2005; Klebanoff et al., 2004; Fehniger and Caligiuri, 2001b; Caroll et al., 2008). A recombinant form of IL-15 [rHuIL-15] is usually expressed in inclusion body and solubilized, refolded and purified Erythropterin within NCI and other divisions of NIH. The purified rHuIL-15 is usually undergoing pre-clinical investigation in preparation for any Erythropterin Phase I clinical study of intravenous administration in patients with refractory metastatic malignant cell malignancy. Measurement of biological activity (potency) of the rHuIL-15 product, and monitoring the stability and lot-lot regularity in biological activity is usually a critical component for product release for clinical investigation studies. For many cytokines, cell proliferation activity on susceptible cells is used as a surrogate potency assay. Available cell-based bioassays for cytokines and the theory and applications of the bioassays are summarized in two review articles (Mire-Sluis et al., 1995; Meager, 2005). The most widely used type of cell proliferation assay is based on the detectible increase or decrease in DNA synthesis as.