The enzyme-linked immunospot (ELISpot) assay is a flexible and sensitive method for investigating the frequency of cytokine-secreting cells within a population. ELISpot is particularly useful for analyzing the immunological functions and responses of peripheral and lymphoid white blood cells, enabling detection of individual cytokine-secreting cells.
The ELISpot assay procedure is very similar to that of a conventional ELISA. Membrane-backed microplates are first coated with the appropriate concentration of capture antibody and allowed to absorb. The unbound antibody is aspirated and the plate is washed. Each well is then filled with a blocking solution and allowed to incubate at room temperature. Alternatively, a variety of ELISpot kits are available with microplates pre-coated with capture antibody and blocked to prevent non-specific protein binding. Cultured secreting cells are added to the prepared microplate wells along with any experimental mitogen or antigen of interest. Cells are maintained for an appropriate incubation period after which they are removed by washing. Secreted molecules remain bound to the capture antibodies in close proximity to the location on the membrane where secreting cells were situated. Enzyme-conjugated detection antibodies and substrate are then added, generating a precipitating product that is visible on the microplate surface.
There are a number of alternative immunoassays available for measuring cytokine secretion in which cytokines can be measured, each with advantages and disadvantages. Enzyme-linked immunosorbent assays (ELISA) are most often used to measure cytokines in serum or plasma, however binding proteins, rapid turnover, and detection difficulties can limit their effectiveness. In situ-hybridization and reverse transcription-PCR can be used to measure cytokine mRNA expression, but these results may or may not reflect true cytokine protein levels. In contrast, ELISpot assays enable sensitive identification of actively secreting cells from a mixed population with limits of detection down to 1 in 100,000 cells.
BioTek provides a full range of automated solutions for conducting ELISpot assays across all commonly used formats. Optimized image acquisition and analysis protocols for the Cytation™ 7 Cell Imaging Multimode Reader deliver accurate and reproducible results. Additionally, integrating the BioTek 405™ TS Washer automates the most labor intensive aspect of the ELISpot workflow, while the BioStack™ Microplate Stacker enables processing up to 50 plates.
Stimulation of Human Peripheral Blood Mononuclear Cells
Human Peripheral Blood Mononuclear Cells (PBMCs) are routinely isolated from blood samples and then used in several fields of research including autoimmune disorders, infectious diseases, vaccine development and cancers. The ELISpot assay monitors ex vivo cellular immune responses to antigenic stimuli. Here we use the Cytation 7 Cell Imaging Microplate Reader in conjunction with Gen5 Microplate Reader and Imager Software to quantitate changes in cytokine secretion in PBMCs using the colorimetric ELISpot assay format. Click here for more.
Automating the Wash Steps of ELISpot Assays
using BioTek’s 405 TS Microplate Washer
Enzyme-linked immunospot (ELISpot) assays are a flexible and powerful tool for investigating secreted molecules from whole cells, particularly for analyzing the immunological functions and responses of peripheral and lymphoid white blood cells. The ELISpot assay can provide information regarding secreted molecules that other techniques cannot. While the analysis of the ELISpot assay can be automated using the Cytation 7 Cell Imaging Multi-Mode Reader, the wash steps inherent in the assay have usually been accomplished manually. Here we describe a convenient and effective method for automating the most tedious aspects of conducting ELISpot assays using the versatile 405 TS Microplate Washer. Click here for more.
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