ELISPOT, a new tool for research and diagnostic

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YOUNG RESEARCHER'S CORNER - SUMMER 2011, by Brigida Immacolata

Dear all, this is my turn to give a contribution to the Young Research Corner and the topic is: ELISPOT, a new tool for research and diagnostic.

The Enzyme-linked immunosorbent spot (ELISPOT) assay was developed by Cecil Czerkinsky in 1983, as a method to monitor immune responses in humans and animals. Similarly to an ELISA immunoassay, it was originally used to count antibody-specific secreting B cells and then to also identify cytokine-producing cells at the single cell level.

Moreover, it allows the detection of low frequencies of cytokine secreting cells. This assay provides evidence of the secretory product of individual activated or responding cells. During the assay, each developing spot represents a single reactive cell that reacts to the substrate and the final number of spots per well provides evidence of how many antigen-specific cells are present in the tested cell population. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information. The good resolution of the number of secreting cells allows the capacity to test also specific cell subsets, often present at lower percentages among the general population.

The ELISPOT assay is more sensitive than tetramers/pentamers stainings or intracytoplasmic stainings for detectable cytokines by cytofluorimetry in which one can have only the idea of how much the specific cytokine is produced by or expressed in a specific cell population, even this population is rare (i.e. antigen-specific response). To over-come the problems of release of the cell product, its dilution in the supernatant and its degradation, ELISPOT assays took advantage on the use of nitrocellulose or polyvinylidene fluoride (PVDF) membranes to capture and identify spots formed by cytokine secreting cells, which directly sit on the membrane. Thus, the cytokine is captured around the secreting cell as it is released where the local concentration of the cytokine is high, before dilution of the cytokine in the supernatant. Moreover, the detection of spots required a short incubation time with the substrate. This makes ELISPOT assays much more sensitive than conventional ELISA measurements.

To detect antibody/cytokine-specific cells, the plates are coated with monoclonal (more specificity) or polyclonal capture antibodies, chosen for their specificity for the analyte to detect. After blocking of aspecific bounds with a serum protein that is non-reactive with any of the antibodies in the assay, the cells of interest are plated at different numbers, along with antigen or mitogen and then placed into incubator for a specified time period. Then, the coated antibody on the membrane captures cytokine/Ig or other cell products secreted by activated cells locally. After washing to remove cells, debris, and media components, a biotinylated polyclonal antibody specific for the compound that will be detected and reactive to a distinct epitope of the target cytokine/Ig is added to the wells and thus is used to detect the captured cytokine/Ig. After washing to remove the excess of un-bound antibody, the detected analyte is visualized with a streptavidin-HRP and a precipitating substrate, revealing a coloured spot (usually a blackish blue) that represents an individual cytokine/Ig-producing cell.
The spots can be counted with an automated reader to capture the microwell images and to analyze spot number and size.

Applications in T or B cells studies offer the possibility to measure the magnitude and the quality of cell immunity at single cell level by detecting individual events of Ag-specific T cells that engage the secretion of cytokines and effector molecules such as GZ-B and or perforin or specific Ig-secreting plasmablasts. Compared to intracytoplasmic stainings, the ELISPOT assay provides information of the frequency and the effector function of T or B cells. Moreover it is possible to use both freshly isolated cells like PBMC or thawed cells, with the final goal to detect how many cells produce a determined compound. Furthermore, it is possible to use the same live cells for other purpose (i.e. FACS staining, cell culture, freezing).

This offers the possibility to test the same cells for different assays, representing a good option for diseases in which the cell number is a critical limiting factor or for patients with a seriously compromised immune system (i.e. HIV or CVID patients).

By Immacolata Brigida

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“A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells.” Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A. J Immunol Methods. 1983 Dec 16;65(1-2):109-21.

“Comparison of a limiting dilution assay and ELISpot for detection of memory B-cells before and after immunisation with a protein-polysaccharide conjugate vaccine in children.” Blanchard-Rohner G, Galli G, Clutterbuck EA, Pollard AJ. J Immunol Methods. 2010 Jun 30;358(1-2):46-55.

“Upregulation of IL-21 Receptor on B Cells and IL-21 Secretion Distinguishes Novel 2009 H1N1 Vaccine Responders from Nonresponders among HIV-Infected Persons on Combination Antiretroviral Therapy.” Pallikkuth S, Pilakka Kanthikeel S, Silva SY, Fischl M, Pahwa R, Pahwa S. J Immunol. 2011 Jun 1;186(11):6173-81.

“Antibody forming cells and plasmablasts in peripheral blood in CVID patients after vaccination.” Chovancova Z, Vlkova M, Litzman J, Lokaj J, Thon V. Vaccine. 2011 May 31;29(24):4142-50.

“Anergic responses characterize a large fraction of human autoreactive naive B cells expressing low levels of surface IgM.” Quách TD, Manjarrez-Orduño N, Adlowitz DG, Silver L, Yang H, Wei C, Milner EC, Sanz I. J Immunol. 2011 Apr 15;186(8):4640-8.