ELISpot versus ELISA: A comparison

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.

**Table. ELISpot and ELISA: similarities and differences**
	ELISpot	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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