Alexander E. Kalyuzhny

Membranes used for western blotting can be also used for ELISPOT, an enzyme-linked immunospot assay, which allows determining frequencies of cytokine-secreting immune system cells. In addition to their high antibody-retaining capacity PVDF and NC membranes provide good support to immune system cells cultured in vitro and do not affect their physiology. ELISPOT assays utilizing membrane-backed microplates are used in many areas of research including vaccine development, HIV research, cancer and infection disease research, autoimmune disease, and allergy research. ELISPOT utilizes the same antibody “sandwich” technique as enzyme-linked immunosorbent assay, but unlike the latter ELISPOT belongs to state-of-the-art techniques when outcome of the assay depends on skills and accuracy of the operator, a thorough selection of matched pairs of capture and detection antibodies, and using appropriate staining reagents. This review covers basics of ELISPOT assay including its immunochemical design, selection of reagents and membrane microplates, and some troubleshooting recommendations.