Method

The EliSpot Method

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- a short introduction to the method

EliSpot (or ElisaSpot, short for Enzyme-linked Immunosorbent Spot 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 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.

AID and the emerging EliSpot field

AID was one of the first companies to offer professionally designed EliSpot readers as well as ready-to-use EliSpot kits. Since 1989 AID develops and manufactures immunoassays and imaging devices to read them. Since 1997, AID has been making EliSpot assay kits and the AID EliSpot Reader System. Assays, hardware and software are original AID developments. The AID EliSpot Assay kits contain a precoated EliSpot plate, a secondary (detection) antibody, an enzyme conjugate and the substrate.

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.

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 immunosorbent spot (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.

We examined IFN-gamma secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-gamma secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

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

The ELISPOT assay is a specific, sensitive, quantitative assay for assessing cell-mediated immune responses to a variety of antigens including HIV-1 peptides. In an IFN-gamma-ELISPOT assay, peripheral blood mononuclear cells (PBMC) from two HIV-1 exposed seronegative (ESN) individuals appeared to respond strongly to an HIV Gag peptide.

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.

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase IFN-gamma production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells.

Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.

Typical images of wells

Example 1: IFN-gamma, human. Although the wells need not be as dark as this, this well represents a good assay. The spots are clearly defined but reflect their origin of diffusion, the rim of the well es clean and the background is even. There are no patterns to the spots.
Example 2: IFN-gamma, human. The PVDF membrane has been rinsed with ethanol to improve coating with the capture antibody.
Example 3: IFN-gamma, human. The PVDF membrane has been coated without pretreatment. Poorly defined spots and a tendency to give a messy rim.
Example 4: B-cell EliSpot often gives huge, imprecisely outlined spots but this is a very nice IgG assay.
Example 5: IFN-gamma, mouse. Poor coating and prolonged cell culture give patches of confluent spots, messy rim and overall questionable count results.