Response surface methodology to determine optimal cytokine responses in human peripheral blood mononuclear cells after smallpox vaccination - PubMed (original) (raw)

Response surface methodology to determine optimal cytokine responses in human peripheral blood mononuclear cells after smallpox vaccination

Jenna E Ryan et al. J Immunol Methods. 2009.

Abstract

Feasibility, amount of sample aliquots, processing time and cost are critical considerations for optimizing and conducting assays for large-population based studies. Well designed statistical approaches that quickly identify optimal conditions for a given assay could assist efficient completion of the laboratory assays for such studies. For example, assessment of the profile of secreted cytokines is important in understanding the immune response after vaccination. To characterize the cytokine immune response following smallpox vaccination, PBMC obtained from recently vaccinated subjects were stimulated with varying doses of live or UV-inactivated vaccinia virus and cultured for up to 8 days. In this paper, we describe a novel statistical method to identify optimal operating conditions for length in culture and virus MOI in order to measure a panel of secreted Th1, Th2, and inflammatory cytokines. This statistical method is comprised of two components. It first identifies a subset of the possible time in culture by virus MOI combinations to be studied. It then utilizes response surface analysis techniques to predict the optimal operating conditions for the measurement of each secreted cytokine. This method was applied, and the predicted optimal combinations of length in culture and virus MOI for maximum vaccinia-specific cytokine secretion were identified. The use of the response surface methodology can be applied to the optimization of other laboratory assays; especially when the number of PBMC available limits the testing of all possible combinations of parameters.

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Figures

Fig. 1

Fig. 1

Vaccinia-specific IFN-γ secretion from PBMC of six optimization subjects. After resting overnight in the presence of 50 IU/mL IL-2, 2×105 PBMC were stimulated in triplicate with either live or UV-inactivated vaccinia virus at an MOI of 0.1. Cell-free supernatants were removed after 3 days in culture at 37 °C and secretion of IFN-γ was determined by ELISA. Bars represent the difference between the median of the stimulated and the median of the unstimulated wells.

Fig. 2

Fig. 2

A). Response surface for vaccinia-specific IL-1β response of PBMC stimulated with inactive vaccinia virus. Panel A depicts the three-dimensional response surface, plotting time and MOI on the log scale, as originally included in the regression analysis. The maximum response value for this surface occurred at a time of 19 h and an MOI of 0.7. B). Response surface for vaccinia-specific IL-1β response of PBMC stimulated with inactive vaccinia virus. The shaded region of Panel B displays all combinations of time and MOI with resulting responses falling within one standard error of the maximum. All such values can be considered statistically equivalent to the maximum. For ease of interpretation, time and MOI values for Panel B are reported in their original (that is, back-transformed) sampling units.

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