ELISPOT Assay as a Tool to Study Oxidative Stress in Lymphocytes

Enzyme-linked immuno spot (ELISPOT) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. We worked out a protocol to study oxidative stress in human peripheral blood lymphocytes by determining their potency to secrete IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha in response to acute treatment with hydrogen peroxide. We show that hydrogen peroxide-induced oxidative stress can cause a ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokines IL-8 and TNF-alpha. However, the number of cells secreting TH2 cytokines IL-4 and IL-5 in hydrogen �peroxide-treated group did not change. It appears that oxidative stress may affect TH1–TH2 cytokine secretion �balance which, in turn, may underlie developments of various pathological conditions. This protocol can be easily modified to study the effects of many other oxidative stress compounds.