The ELISpot (Enzyme-Linked ImmunoSpot) assay is a highly sensitive immunoassay that measures the frequency of individual cells secreting a specific molecule, typically cytokines or antibodies, in vitro. It’s widely used for detecting and quantifying antigen-specific T cells or antibody-secreting cells.
The ELISPOT assay begins by coating plates with a capture antibody. These plates are similar to ELISA plates but have a nitrocellulose or PVDF membrane. The coating process is followed by an incubation period, allowing the antibodies to bind to the membrane.
A blocking buffer, usually BSA or other proteins, is added to the plates. It’s incubated for 1-2 hours at room temperature. This prevents non-specific binding on the membrane.
Cells are then added to the plates. These can be antigen-specific T cells, B cells, or other immune cells. The plates are incubated for 18-24 hours at 37°C in a CO2 incubator. During this time, the cells secrete the target cytokine or antibody.
After incubation, the plates are washed. This step removes cells and unbound substances. A biotinylated detection antibody specific to the target is added next. Following another wash, a streptavidin-enzyme conjugate and a substrate are applied. This produces visible spots indicating the presence of the target molecule.e that the enzyme converts to a visible spot.
The final step involves counting the spots. Each spot represents a single secreting cell. This process is done using a microscope or an automated reader. The count provides quantitative data on the cells secreting the target molecule. This is vital for evaluating immune responses or vaccine efficacy.
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Automating the ELISpot workflow introduces a high degree of precision, efficiency, and consistency, essential for high-throughput screening and large-scale immunological studies. This transformation from manual to automated processes not only saves significant time but also reduces the variability often introduced by manual handling.
ELISpot specializes in detecting specific cellular responses at the single-cell level. It offers high sensitivity and quantifies individual secreting cells. This makes it perfect for detailed functional analysis with minimal sample needs. On the other hand, ELISA measures the total concentration of cytokines or antibodies in a sample. It is better suited for broad quantification without cellular specificity. Thus, ELISpot is the choice for precise cellular studies, while ELISA is preferred for assessing overall protein levels in samples.
