Elispot Assay Services
- Quantitation of antigen specific cell mediated immune response
- Ability to detect low precursor frequency CD4+ T cells and CD8+Tcells
- Customized development and validation services
- Evaluate memory response to whole recombinant proteins/peptides derived from proteins
- Epitope map the antigenic regions of a protein
AcuImmune’s Elispot Assay Advantages
- Capable of handling both large and small scale research projects.
- Quick turn-around time from project request to processed data.
- Competitive pricing.
- Save precious resource; reduce cost and labor
- Ensure use of highest quality kits from reputable manufacturers
- Capable of handling cryopreserved samples from multiple species: human, canine, rodent, and non human primates
- Optimized cell thawing and assay protocols
- Complete analysis and interpretation of data
Elispot assays
The Elispot assay was developed from a modified version of the ELISA immunoassay. Elispot assays were originally developed to enumerate B cells secreting antigen-specific antibodies, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the Elispot assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the Elispot assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.
By virtue of exquisite sensitivity of the Elispot assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part because the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes Elispot assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection (Source: Wikipedia).
Procedure
As noted above, the Elispot assays employ a method very similar to the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal (preferred for greater specificity) or polyclonal capture antibody that is coated aseptically onto a PVDF (polyvinylidene fluoride) -backed microplate. These antibodies are chosen for their specificity for the analyte in question. The plate is blocked, usually with a serum protein that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities, along with antigen or mitogen, and then placed in a humidified 37°C CO2 incubator for a specified period of time.
Cytokine (or other cell product of interest) secreted by activated cells is captured locally by the coated antibody on the high surface area PVDF membrane. After washing the wells to remove cells, debris, and media components, a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells. This antibody is reactive with a distinct epitope of the target cytokine and thus is employed to detect the captured cytokine. Following a wash to remove any unbound biotinylated antibody, the detected cytokine is then visualized using an avidin-HRP, and a precipitating substrate (e.g., AEC, BCIP/NBT). The colored end product (a spot, usually a blackish blue) typically represents an individual cytokine-producing cell. The spots can be counted manually (e.g., with a dissecting microscope) or using an automated reader to capture the microwell images and to analyze spot number and size.
Links
Animation of the ELISpot technique
Examples of patterns formed in the course of an ELISPOT assay
References
Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A (1983). “A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells”. J Immunol Methods 65 (1-2): 109–21. doi:10.1016/0022-1759(83)90308-3. PMID 6361139.
Moodie,Z.,Price,L. Gouttefangeas,C,Mander,A, Janetzki,S, Loer,M,Welters,MJP, Ottensmeier, Van der Burg,SH, Britten,CM(2010). “ Response definition criteria for ELISPOT assays revisited.” Cancer Immunol Immunother. 2010 October; 59(10): 1489–1501
Gill DK, Huang Y, Levine GL, Sambor A, Carter DK, et al. (2010) Equivalence of ELISpot Assays Demonstrated between Major HIV Network Laboratories. PLoS ONE 5(12): e14330. doi:10.1371/journal.pone.0014330
Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials. Moodie Z, Huang Y, Gu L, Hural J, Self SG. J.Immunol Methods. 2006 Aug 31;315(1-2):121-32. Epub 2006 Aug 15.