Quantitation of T-cell cytokine responses by ELISA, ELISPOT, flow cytometry and reverse transcriptase-PCR methods

In this chapter, we have described the protocols for Thl and Th2 cytokine-specific ELISA, ELISPOT, intracellular cytokine and quantitative RT-PCR assays for mice. These four assay systems allow detection of different stages of cytokine production including secreted cytokine, cytokine-producing cells and cytokine-specific mRNA, respectively. Although each assay has unique advantages for the detection and quantitation of Thl and Th2 cytokines at three distinct levels (e.g. protein, mRNA and cell) in the murine model, the use of individual assays in a separate manner may often not be sufficient for a thorough and accurate determination of the profile of Thl or Th2 cytokine expression. For instance, when the levels of cytokine-specific mRNA are measured by quantitative RT-PCR only, the information concerning the biological activity of analysed cytokine is unknown. Further, examination of secreted cytokines in culture supernatants by ELISA may only detect residual cytokines produced by CD4+ T cells in vitro, since it is possible and even likely that secreted cytokines may be immediately taken up by neighbouring cells in the culture. To this end, it is best to perform at least two different cytokine assays to confirm the results describing the profile of Thl and Th2 cytokine responses induced by mucosal and/or systemic immunization. An ideal situation would be to use these three different assays for the elucidation of Thl and Th2 responses.