Cytokine secretion assay

Recently, Sen et al. (2001) have described methods for generating highly effective HER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2 bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodies were conjugated and used to arm T cells that were subsequently tested for binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively kill HER-2-positive breast cancer cells (MCF-7). 

We have run a screening campaign in which the activation of GR was measured by its translocation to the nucleus using the DiscoveRx PathHunter system. To complement the results of that assay and evaluate the function of GR as a result of compound incubation, we ran a cytokine secretion assay in which putative GR agonists were incubated with primary human monocytes prior to challenge with lipopolysaccharide (LPS) (Patel et al 2009). Treatment with a GR agonist such as dexamethasone will prevent the LPS-evoked secretion of inflammatory cytokines such as TNFa. 

A sample protocol for the 384-well plate cytokine secretion assay is listed below ... 

Patel, A. et al. 2009. A combination of ultra high-throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonists. Anal. Biochem. 385, 286-302. 

Tanguay, S., and Killion, J.J. (1994) Direct comparison of ELISPOTand ELISA based assays for detection of individual cytokine secreting cells. Lymphokine and Cytokine Research, 13, 259 263. 

Fig. 2. A -THC enhances CB2R+ + splenocyte IL-2 cytokine secretion, but not CB2 R splenocyte IL-2 secretion. Splenocyte proliferation was determined by BrdU incorporation assay as described in the text. Cells were plated at 2 xlO cells/0.1 tnL/weU in complete media. Cells were treated with 2.5 itg/mL ConA and the indicated A -THC concentration for 72 h. Data are experiments and are expressed as the mean of quadruplicate samples standard deviation. Vehicle is absolute ethanol.

Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

In addition to above-mentioned cytokines, IRIV were also shown to induce expression and secretion of various chemokines, such as IP-10, MIG, and Rantes (Schumacher R, unpublished). Secretion of chemokines is important for the recruitment of immune cells however, the relevance of this in vitro finding has not been addressed by migration assays or by in vivo studies so far. 

While in most instances the RCA microarray immunoassay results were comparable to published performance reports on commercial ELISA (Quan-tikine, R D Systems), there were notable exceptions in which ELISA appeared to out-perform the microarray by 10- to 50-fold sensitivity. Such differences may be due to variations in binding affinities exhibited by the particular capture antibodies employed in the assay. In studies involving lipopolysaccharide (LPS)-induced secretion of cytokines from human... 

Anti-arthritic effect. Oral administration of AJA, a cannabinoid acid devoid of psychoactivity, reduced joint tissue damage in rats with adjuvant arthritis. Peripheral blood monocytes (PBM) and synovial fluid monocytes (SFM) were isolated from healthy subjects and patients with inflammatory arthritis, respectively, treated with AJA (0-30 mM) in vitro, and then stimulated with lipopolysaccharide. Cells were harvested for messenger RNA (mRNA), and supernatants were collected for cytokine assay. Addition of AJA to PBM and SFM in vitro reduced both steady-state levels of interleukin-ly (IL-ly) mRNA and secretion of IL-ly in a concentration-dependent manner. Suppression was maximal (50.4%) at 10 mM AJA (p < 0.05 vs untreated controls, n = 7). AJA did not influence tumor necrosis factor-a (TNF-a) gene expression in or secretion from PBM . 

The ELISpot assay has been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich ELISA technique. A monoclonal antibody specific for the cytokine is precoated onto a microplate. Cells are pipetted into the wells of the microplates. During the incubation period, the immobilized antibody (in the immediate vicinity... 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

IFN-y is a potent immunostimulatory and anti-viral cytokine. The frequency of specific IFN-y-secreting cells stimulated by liposomal formulations exclusively containing the CTL epitope NS 18 51 or combinations with CpG, respectively, was evaluated by ELISPOT assay. Two weeks after three immunizations, high numbers of specific IFN-y-secreting cells in mice immunized with liposomes containing NS1851 (-0.2% of total spleen cells) were detected which further increased in mice immunized with liposomal formulations containing CpG (-0.7% of total spleen cells) (Fig. lb). 

Enzyme-linked immunosorbent assays (ELISAs) have long been used to quantify cytokines and other proteins that are secreted from cells. ELISAs enable the user to assay multiple samples, to obtain reproducible quantitative results, and to design studies with quantifiable endpoints. Intracellular activities intimately involved in apoptosis, such as control of mitochondrial permeability to holocytochrome c and activation of a specific caspase, are quantified by the ELISAs described in this chapter. The cytochrome c ELISA is generally used to quantify the activities of the proteins belonging to the Bcl-2 family. The active caspase ELISA quantifies a specific active caspase among a background of latent caspase and other active caspases. 

Most of the initial studies used in vitro proliferation or IL-2 production assays to demonstrate induction of T cell anergy. In some cases, T cell anergy was also shown to operate in vivo [30, 38], but not in others. Thus, in vivo SAg challenge of anergic mice induced secretion of IL-2 and other cytokines in vivo [39, 40], Further, CD4+ T cells that were anergic when exposed to the antigen in vitro were able to induce efficient B cell responses in vivo [13, 41, 42],... 

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