Enzyme-linked immunospot assay for detection of thioredoxin and thioredoxin reductase secretion from cells.

Oxidative stress response was determined in this study by enzyme-linked immunospot (ELISpot) assays for thioredoxin (Trx) and Trx reductase (TrxR). On exposure to oxidative stress, cells can launch a variety of defense mechanisms, including release of antioxidant proteins. The Trx system, consisting of Trx, TrxR, and NADPH, constitutes one of these cellular defense systems for maintenance of a healthy reduction-oxidation (redox) balance. Trx and TrxR are rapidly upregulated and released from monocytes, lymphocytes, and other normal and neoplastic cells on exposure. Secreted Trx and TrxR have proved to be eminent indicators of oxidative stress. Trx is a small, 12-kDa protein released through a leaderless pathway, whereas TrxR, which is a 116-kDa selenoprotein and required for regeneration of Trx, is secreted through the Golgi pathway. In this chapter we present a detailed laboratory bench protocol for enumeration of single cells secreting redox-active Trx and TrxR after oxidative stress exposure. Physiological stimuli (such as interferon gamma, lipopolysaccharide, interleukin 1, and CD23 ligation; and phorbol 12-myristate 13-acetate and ionophore) as well as UV light and hydrogen peroxide were used to generate oxidative stress, and some are presented in detail. The protocol includes a description of cell isolation, preparation, handling, and development of ELISpot plates, troubleshooting notes, presentation of results, statistical evaluation, and comments on alternative sources of materials and manufacturer Web addresses. We concluded that the ELISpot assay is a useful method for detection of single cells secreting the redox-active proteins Trx and TrxR after oxidative stress exposure.