Automated ELISA Spot Counter
Introduction to EliSpot
The Enzyme-linked immunosorbent spot (ELISPOT) assay is a common method for monitoring immune responses in humans and animals. The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. Either a monoclonal or polyclonal capture antibody 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.
Equipment: AID iSpot reader
AID EliSpot reader interprets any type of Elispot plate, including all brands of membrane type plates, ELISA-style plates and low volume plates. Software and hardware are fully compliant with 21CFRpart11, GMP/GLP and CE.
The AID EliSpot Reader simultaneously takes images, autocenters the well and counts according to the user’s settings. Data acquisition is fully automatic. Counting results can be exported to txt, dbase, Excel, PowerPoint, LIMS, AIDiagnostics or EliStat. Processing time including saving and data export is less than 2 minutes per plate.
In addition, the iSpot Reader simultaneously allows for 2 or even 3-colour FluoroSpot analysis. A simple “two-click switch” between fluorescent and enzymatic mode, without the need of hardware changes, is enough to come from one mode to the other.
You can operate this device easily, with or without help from facility personnel. Upon request, we provide a 1 hour training session for the AID iSpot reader.
Please contact us if you want to do Elispot and/or use the automated iSpot reader.