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Sunday, October 27, 2013

ELISPOT - Procedure / Protocol, Advantages, ELISA Vs ELISPOT


The Enzyme Linked Immunospot (ELISPOT)  technique was developed by Cecil Czerkinskdy in 1983. ELISPOT is used for the detection of secreted proteins, such as cytokines and growth factors. ELISPOT is primarily used in immunology research in the following areas:
  • Transplantation – prediction of infectious risk
  • Vaccine development (IFN╬│)
  • Th1/Th2, T-cell regulation analysis
  • Monocyte and Dendritic cell analysis
  • Autoimmune disease
  • Cancer – tumor antigens
  • Allergy
  • Viral infection monitoring and treatment

Elispot assay

In this technique, as in ELISA 96-well plate is used but the plates will have PVDF or nitrocellulose membrane at the bottom of the well. The membranes are coated with primary antibody and the cells are grown on that wells. As the cells settle on to the membranes are secrete proteins, the protein of interest will bind to the coated primary antibody. The protein of interest can be detected using a secondary antibody.The protein will be seen as a spot of color. One spot corresponds to one cell.Using computer softwares the spots can be scanned and analyzed.

Advantages of  ELISPOT
  • Sensitive assay
  • Functional assay
  • Adaptable
Difference between ELISA and ELISPOT (ELISA Vs ELISPOT)

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).

ELISPOT will yield information about number of cells secreting cytokine of interest (e.g. n/1 million cells)

ELISA will yield information on the Concentration of the cytokine of interest produced by all cells in the culture (e.g. pg/1 ml of supernatant)

ELISA gives you information about how much cytokine is produced and secreted at a given point of time, but no information about how many cells are secretors of the cytokine.

In ELISPOT there is possibility of detecting parallel secretion of 2 proteins by the same cell

In ELISA it is not possible to detect secretion of 2 proteins by the same cell parallely.

Reference

Cellular Technology Limited - Technical Resource
Abcam - Technical Resources
Other Internet Resources