• EliSpot- a short introduction to the method
• AID and the emerging EliSpot field
• Publications with regard to the EliSpot method
• Typical images of wells

EliSpot (or ElisaSpot, short for Enzyme-linked ImmunoSpot Assay) originally was developed as a method to detect antibody-secreting B-cells. Later the method was adapted to determine T-cell reaction to a specific antigen, usually represented as number of activated cells per million. Most researchers use Interferon gamma (IFN-g) production as a read-out for activation of single cells.

Elispot serves to determine cytotoxic T-cell activity in viral (HIV/Aids, Hepatitis) or tumor related studies or to determine Th1-Th2 balance. EliSpot Assays are routinely performed in 96-well micotiter plates with a membrane bottom to each well. The method is not new but has only in recent years been made usable with the development of EliSpot analyzers (analysers), imaging devices that take a picture of a single well of the EliSpot plate and enumerate the spots.

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Kits are available for most human cytokines as well as for some animal parameters. Like all other AID products, Elispot Assay Kits are manufactured under strict ISO-certified quality control to insure standardized response. Kits are also customized on demand. The classic AID EliSpot Reader System has been the choice of many distinguished research labs on all continents. Compact , robust and user-friendly, it is one of the most successful developments in this area. We are particularly proud to equip the IAVI clinical trials for HIV vaccine delopment with several of our AID EliSpot Reader HR.

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Publications with regard to the EliSpot method

A selection of papers we found of technical interst to the EliSpot method (if you have published or find a paper we should include in this section, please email!).

J Immunol Methods. 2002 Feb 1;260(1-2):157-72. A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays.
Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, Cox JH. The US Military HIV Research Program, Suite 200, 13 Taft Court, Rockville, MD 20851, USA.

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8-11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals.

J Immunol Methods. 2004 Feb;285(1):89-92. Poorly soluble peptides can mimic authentic ELISPOT responses. Karlsson RK, Jennes W, Page-Shafer K, Nixon DF, Shacklett BL.
Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141, USA

Analysis of this peptide revealed that it was incompletely dissolved and induced non-specific spot formation, even in the absence of cells. In subsequent experiments, the peptide was found to interact with avidin and the ELISPOT membrane. Filtering the peptide prevented non-specific spot formation. These findings underscore the need for appropriate controls and proper peptide preparation in order to reduce the risk of false-positive ELISPOT responses. J Immunol Methods. 2002 Dec 1;270(1):99-108.

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15--the Amplispot assay. Jennes W, Kestens L, Nixon DF, Shacklett BL. Gladstone Institute of Virology and Immunology, University of California-San Francisco, San Francisco, CA, USA.

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