In it's main principle, the enzyme-linked immunospot assay (ELISpot) resembles the enzyme-linked immunosorbent assay (ELISA). In both techniques pairs of antibodies are used: primary antibodies (catching antibodies) and secondary antibodies (detecting antibodies). Beside this, however, there are more differences than similarities. Thanks to these differences, ELISpot overcomes certain limitations of ELISA. Moreover, combining both techniques in one experiment supplies additional information, e.g. it is possible to calculate the mean production of a cytokine by single stimulated cell (e.g. pg per cell).
From the user's point of view, the main advantage of ELISpot is sensitivity of the method. ELISpot allows for detection of a single cell that secrets a protein of interest (cytokine, effector protein, receptor, surface marker, antibody) among 1,000,000 cells in the culture. In ELISA, detection of proteins secreted by a single cell is impossible. Sometimes, cytokine production by hundreds of stimulated cells remains undetected because of the insufficient sensitivity of ELISA and/or the ongoing consumption of the cytokine by cultured cells. In many situations, detection of interleukin 2 (IL-2) in cell cultures with ELISA appears impossible, because cultured cells instantly consume the secreted IL-2. In contrast to ELISA, ELISpot is not affected by cytokine consumption, and thus very well suitable for the detection of cells secreting IL-2. The table compiles similarities and differences between ELISpot and ELISA.
|Result:||Number of cells secreting cytokine of interest (e.g. n/1 million cells)||Concentration of the cytokine of interest produced by all cells in the culture (e.g. pg/1 ml of supernatant)|
|Bias due to consumption of protein of interest by cultured cells:||NO||YES|
|The principle of protein detection:||Immunoenzymatic reaction||Immunoenzymatic reaction|
|Primary antibodies:||Culture plate coated before starting the culture||Coating takes place in additional plate afte the culture|
|Secondary antibodies:||Culture plate coated after finishing the culture||Coating takes place in additional plate after the culture|
|Possibility of detecting parallel secretion of 2 proteins by the same cell:||YES (Dual ELISpot)||NO|
|Colour reaction:||Alkaline Phosphatase (ALP) + colour substrate or Horseradish Peroxidase (HRP) + colour substrate||Alkaline Phosphatase (ALP) + colour substrate or Horseradish Peroxidase (HRP) + colour substrate|
|Detection and colour reaction:||In the cell culture (ELISpot) plate||In the supernatant in a separate ELISA plate|
|Stopping the colour reaction:||By washing with water and drying the plate||By changing pH (adding H2SO4) in the liquid phase|
|Final analysis in:||Dry phase||Liquid phase|
|Detection device:||ELISpot reader/scanner (camera + visual analysis software)||ELISA reader (photometer)|
|Acquisition of numerical data:||Computerised visual analysis (1 spot = 1 cell)||Measurement of lightwave extinction in the solution and calculation of concentration from calibration curve|
|Possibility of sending the plate by mail for analyses:||YES||NO|
|Storage time with possibility of (re-) analysis:||Weeks - months||Hours|
|Attention: antibodies used for ELISA are not always suitable for ELISpot. Usage of antibody pairs specifically assigned for ELISpot is advisable (most antibody producers offer such pairs). Also, not all colour substrates for ELISA would function properly in ELISpot.|
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