A Solid-phase Enzyme-linked Immunospot (ELISPOT) Assay for Enumeration of Specific Antibody-secreting Cells

A solid-phase enzyme-linked immunosorbent assay (ELISPOT) is described for enumeration of cells secreting specific antibody. Spleen cells from immunized mice are incubated in antigen-coated polystyrene plates. After removal of the cells, bound antibodies are demonstrated by means of an immunoenzyme procedure in which enzyme-substrate reactions are performed in agarose. Dark-brown circular zones (spots), localized in areas of the dish where antibody production has occurred, are enumerated with the naked eye. Spectrophotometric estimation of enzyme-bound activity may be performed by substituting the gel for a liquid buffer, allowing accurate estimation of the total amount of secreted antibody. Versatile, sensitive and very easy to perform, this new assay provides a useful alternative to conventional plaque-forming cell assays.