Elispot assay ( assay to determine cytokine producing cells)

Learn that ELISPOT is an immunoassay used to detect and count individual cells secreting specific cytokines in patient samples like plasma or serum. Understand how it differs from ELISA, which detects antigens or antibodies in bulk, whereas ELISPOT identifies the actual cytokine-producing cells.

Coat the wells of a microtiter plate with antibodies specific to the cytokine you want to detect. These antibodies will capture the cytokines secreted by the cells in the sample.

Add the patient sample containing cytokine-secreting cells to the antibody-coated wells. The cells will secrete cytokines that bind to the capture antibodies coating the well surface.

Perform a wash step to remove all unbound cells and unnecessary substances, leaving only the cytokines bound to the capture antibodies in the wells.

Add secondary antibodies conjugated with enzyme that bind to the captured cytokines. These enzyme-linked antibodies will enable detection of the cytokine-cytokine-antibody complexes.

Perform another wash to remove unbound secondary antibodies, then add substrate to trigger a chromogenic reaction. The enzyme produces colored spots at each location where a cytokine-producing cell was bound.

Count the number of spots that developed in the well; each spot represents one cytokine-producing cell. The spot number is proportional to cell density and allows quantitative analysis of immune responses.

Compare spot numbers between samples to assess the strength of immune responses, such as determining Th17 cell production in candida-infected versus uninfected individuals, or Th1 response in viral infections. Use these comparative analyses to evaluate individual variation and immune status.