Jennifer R Harrington looks at a line of kits which have been designed to detect individual cells secreting specific human and mouse cytokines.
The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies. 1,2 This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens. 3,4 ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbant assay (ELISA) technique.
R&D Systems has developed a line of cytokine-specific human and mouse ELISpot kits. The basic principle and kit components of these ELISpot kits are outlined in Table. 1.
A monoclonal antibody specific for the cytokine of interest has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are then pipetted into the wells and the microplate is placed into a humidified 37°C CO2 incubator for a specified period of time.
During this incubation period, the immobilised antibody in the immediate vicinity of the secreting cells binds secreted cytokine. After washing away cells and any unbound substances, a biotinylated polyclonal antibody specific for the cytokine of interest is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added.
Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is then added for colour development. Blue-black coloured precipitate forms at the sites of cytokine localisation and appears as spots, with each spot representing an individual cytokine-secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.
The human IFN-g ELISpot assay is designed for the detection of IFN-g secreting cells at the single cell level and can be used to quantitate the frequency of IFN-g secreting cells (see Fig. 1). ELISpot assays are well suited for monitoring immune responses to various stimuli, treatments and therapies. They have also been used for the quantitation of antigen-specific CD4 and/or CD8 T cell responses.
Other methods used for assessing antigen-specific T cell responses, such as the chromium release assay with quantitation by limiting dilution, are tedious and require previous in vitro expansion of T cells. These assays are typically not suitable for measuring infrequent T cell responses that occur at less than 1 in 1000. ELISpot assays are highly reproducible and sensitive, and thus can be used to measure responses with frequencies well below 1 in 100000. ELISpot assays do not require prior in vitro expansion of T cells and are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in areas as diverse as antigen recognition, vaccine development, and monitoring of various clinical trials.
ENQUIRY No 49
Jennifer R Harrington is with R&D Systems Inc, Minneapolis, MN, USA. www.rndsystems.com
1. Russell, S M et al. (1993) Science 262:1880. 2. Murata, T et al. (1999) Int. J Hematol. 69:13. 3. Aman, M J et al. (1996) J. Biol. Chem. 271:29265. 4. Pan, P-Y & P Rothman (1999) Curr. Opin. Immunol. 11:615.
