ELISpot

Various assays, including ELISA, immunoblots, and chamber ELIspot, have been employed for demonstrating polyreactivity of preimmune antibodies in both mice and humans (Temynck and Avrameas, 1986 Quan et al., 1997 Klimman, 1994). In a complex biological matrix, polyreactive antibodies are bound by components of the matrix, leaving... 

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

Sandra J. Rosenthal and David W. Wright, 2005 302 Handbook of ELISPOT Methods and... 

VaqeranoJE, Peng M, Chang JW, et al. Digital quantification of the enzyme-linked immunospot (ELISPOT). Biotechniques (1998) 25 830-836. 

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... 

Using the ELISA (with avidin-biotin bridges) and ELISPOT methods a stimulating effect of selected probiotic bacteria (Bifidobacterium longum, B animalis, Lactobacillus easel, L. salivarius) on the immune system of the rat and mouse has been demonstrated (higher level of specific as well as total IgGa and IgA content) (Nagy et al., 2002). 

The above analytical solutions are used in the ELISPOT method, dot-bolt, and immunoblotting (Masseyeff et al., 1993 Bednarski and Reps, 2003 Jedrychowski, 2003) and in analytical systems used for clinical diagnostic purposes (Clinical Laboratory International, 2005). 

The ELISPOT method can be commonly applied to determination of metabolites (e.g., cytokines) produced by cells in minute quantities. 

Sutton IJ, Steele J, Savage CO, Winer JB, Young LS. An interferon-gamma ELISPOT and immunohistochemical investigation of cytotoxic T lymphocyte-mediated tumour immunity in patients with paraneoplastic cerebellar degeneration and anti-Yo antibodies. J Neuroimmunol 2004 150(l-2) 98-106. 

Evaluating the immune response to the vaccine at the same time as evaluating toxicity is recommended in a 2003 WHO guideline (Table 31.1). This makes good sense because any toxicity observed with vaccines is frequently related to the immune response, and correlation of these two endpoints provides a fuller explanation of the test results. Assays to measure the immune response include those which measure antigen-specific antibody responses (e.g., ELISA) and cell-mediated responses (e.g., y-interferon—ELISPOT). Developing these... 

Elispot The use of membranes to measure cells secreting a specific product such as an... 

Sandra J. Rosenthal and David W. Wright, 2005 302. Handbook of ELISPOT Methods and Protocols, edited by Alexander E. Kalyuzhny, 2005 301. Ubiquitin-Proteasome Protocols, edited by Cam Patterson and Douglas M. Cyr, 2005 300. Protein Nanotechnology Protocols,... 

We do not describe in details the immunological methods (immunization, Gr release assay, ELISPOT, ELISA and flow cytometry) used for the analysis of the immune responses induced by the liposome vaccines (Subheading 3.2). For comprehensive information, we refer to our publications (21-23) and to the related literature. 

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. 

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