It is possible to combine various separation and detection procedures for identification and analysis of Ags and for evaluating the expression of molecules by single cells. Immunoblotting can also be used to assay for the presence of molecules in a mixture as described for the sandwich ELISA. This has now been extended for analysis of products of single cells. For example, to assay for production of a cytokine, Ab to the cytokine is coated onto the nitrocellulose ‘floor’ of a special culture well, the unbound Ab is washed off, and cells are then plated on top of this Ab. After incubation, an enzyme-linked Ab to a different determinant on the cytokine is added, followed by washing and substrate addition. Wherever a cell produced the cytokine, it will be captured by the first Ab and will then be detected by the second Ab and its conversion of substrate, forming a colored spot on the nitrocellulose (hence the name ELISPOT assay). The nature of the cell producing the cytokine can also be determined by flow cytometry after staining the cells with a fluorescent-labeled cell-type-specific Ab (e.g. anti-CD4 for T helper cells) and an anti-cytokine Ab labeled with a different fluorochrome.
