Ultrasensitive ELISA for measurement of human cytokine responses in primary culture.

ELISAs offer excellent specificity and, once fully optimized, sensitivity that rivals that of bioassays. The major variables that need to be experimentally determined when developing an ELISA are the optimal number of fresh cells required per well, the optimal antigen concentrations for stimulation, period of culture, and the anticipated intensity of the response. In this chapter, we review the major factors to be considered in the development and application of ultrasensitive ELISAs to the analysis of human immune responses. We specify the conditions we have found to be optimal for quantifying a number of cytokines of demonstrated relevance to human immune regulation and discuss the major pitfalls inherent in this approach.

Systemic chemokine and chemokine receptor responses are divergent in allergic versus non-allergic humans.

T(h)1- and T(h)2-polarized human T cell clones display distinct patterns of chemokine receptor expression and selective chemokine responsiveness in vitro. We hypothesized that natural exposure to environmental grass pollen would induce differential systemic chemokine and chemokine receptor expression patterns in individuals with allergic rhinitis compared to healthy controls with type 2- and type 1-dominated responses to allergen respectively. To this end, we compared chemokine receptor expression on peripheral blood T cells directly ex vivo and plasma chemokine levels between these two groups of study participants prior to and during the grass pollen season. T(h)1-associated CXC chemokine receptor (CXCR) 3 was strongly expressed on >50% CD4(+)/CD45RO(+) cells of all subjects. When examined longitudinally, CXCR3 expression increased over the grass pollen season (P < 0.0001), solely in non-allergic subjects. In contrast, for both allergic and non-allergic subjects, CC chemokine receptor (CCR) 5 (T(h)1-associated) and CCR3 (T(h)2-associated) were weakly expressed on <10% of CD4(+)/CD45RO(+) cells both prior to and during the grass pollen season. Type 1 chemokines CXC chemokine ligand (CXCL) 9 and CXCL10 (monokine induced by IFN-gamma and IFN-gamma-inducible protein of 10 kDa: CXCR3 ligands), and type 2 chemokines CC chemokine ligand (CCL) 11 (eotaxin: CCR3 ligand), CCL17 (thymus and activation-regulated chemokine: CCR4 ligand) and CCL22 (monocyte-derived chemokine: CCR4 ligand) were readily detectable in the plasma of most participants. Systemic CXCL9 levels decreased from pre- to grass pollen season in allergics (P < 0.05), whereas CCL17 decreased in non-allergics (P < 0.05) over the same period. Taken together, these longitudinal data suggest a systemic shift to more intensely type 1-dominated responses in non-allergic individuals and, conversely, to more type 2-dominated responses in allergic individuals upon natural re-exposure to grass pollen.