ELISPOT assay

Stott, D. I. (2000) Immunoblotting, dot-blotting, and ELISPOT assays methods and applications. J. Immunoassay 21, 273-296. 

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... 

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). 

Fig. 1. CTL responses (a) and IFN-y production (b) after s.c. injection of NS1851 liposomes (-130pg peptide three times every 2 weeks) with or without immunostimulatory CpG (250 nmol) or as controls the peptide in saline solution. The CTL response was analyzed 2 weeks after the last injection and measured in a standard Cr release assay after 5 days of re-stimulation in vitro. Black squares represent the response induced by liposomal formulations and open circles the response of the control peptide. IFN-y production was measured by ELISPOT assay after stimulation with peptide pulsed cells...

FIGURE 13.1 Principle of ELISPOT assay. Source Reprinted with kind permission of Springer Science + Business Media from Ref. [40],... 

Czerkinsky, C.C., Nilsson,L.A., Nygren,H., Ouchterlony, O., and Tarkowski, A. (1983) A solid phase enzyme linked immunospot (ELISPOT) assay for enumeration of specific antibody secreting cells. Journal of Immunological Methods, 65, 109 121. 

Czerkinsky, C.C., Andersson, G., Ekre, H.P., Nilsson, L.A., Klareskog, L., and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma interferon secreting cells. Journal of Immunolog ical Methods, 110, 29 36. 

Okamoto, Y., Murakami, H., and Nishida, M. (1997) Detection of interleukin 6 producing cells among various organs in normal mice with an improved enzyme linked immunospot (ELISPOT) assay. Endocrine Journal, 44, 349 355. 

Pass,H.A., Schwarz, S.L., Wunderlich, J.R., and Rosenberg, S.A. (1998) Immunization of patients with melanoma peptide vaccines immunologic assessment using the ELISPOT assay. The Cancer Journal from Scientific American, 4, 316 323. 

Cox, J.H., Ferrari, G., and Janetzki, S. (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods, 38, 274 282. 

As a first important result, these trials confirmed the excellent safety profile of completely synthetic peptides in general, and poly-L-arginine in particular. Furthermore, several important lessons regarding the activation of human T cells were learned in both studies, T cell responses were assessed using [ H]-thymidine prohf-eration and IFN-y ELIspot assays, and fluorescence-activated cell sorting (FACS). 

Lakew M, Nordstrom I, Czerkinsky C, Quiding-Jarbrink M. Combined immunomag-netic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells. J Immunol Methods 1997 203 193-198. 

Monitoring of Cytokines from T Cells and the ELISPOT Assay... 

Granzyme B ELISPOT Assay for the Detection of Drug-Reactive T Cells... 

A highly sensitive ELISPOT assay has been developed for granzyme B but, unlike the IFN-y ELISPOT assay, it directly measures the release of a cytolytic protein making it a more direct measure of antigen-specific cytotoxic T lymphocyte lytic activity. 

ELISPOT assays for cytokines such as IFN-y appear to be potentially promising techniques to aid the diagnosis of delayed reactions to CSs. 

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