Abstract
Membranes are widely used as protein blotting matrices for a large variety of research applications including western blotting and enzyme-linked immunospot assay (ELISPOT). The largest advantage of using membranes versus solid plastic support is the porosity of membranes allowing for immobilization of high concentrations of proteins and antibodies which, in turn, increases the sensitivity of detection. Similar to plastic surfaces, polyvinylidene difluoride (PVDF) and nitrocellulose membranes create good microenvironment for live cells cultured in vitro and do not interfere with cellular physiology. It appears that PVDF-backed microplates are a golden standard for ELISPOT assays: such plates are inexpensive, easy to use and after assay development, membranes can be removed from the plates and archived. Given the convenience and reliability of membrane microplates, they are widely used in ELISPOT assays for basic research and clinical trials. The ELISPOT assay is an antibody "sandwich" technique aimed at trapping cell-secreted molecules between capture and detection antibodies, followed by either chromogenic enzymatic or fluorescence detection. This review covers the principles of the ELISPOT assay on membrane microplates including single-color and two-color detection techniques with the emphasis on assay design, choosing membrane microplates, and troubleshooting protocols.
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