A cell-detachment solution can reduce background staining in the ELISPOT assay.

Enzyme-linked immunospot (ELISPOT) assays are widely used as a technique that allows determining the frequency of cytokine-releasing cells. Colored spots appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. Porous membranes are used in ELISPOT plates to provide support for growing cells, thus making it difficult to remove them by washing. Cells that have adhered to the membrane may be stained nonspecifically, producing a background and then counted as specific spots. We have tested a cell detachment reagent, Accumax, and found that it may be used to remove a large number of cells adhered to the microplate membranes. Accumax was tested in 16 different ELISPOT assays, including human interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-13, IL-1beta, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha; mouse IL-4, IL-6, IFN-gamma, and TNF-alpha; rat IL-2 and IFN-gamma; and canine IFN-gamma. Accumax was found to be compatible with human IL-13, IL-1beta, IL-2, IL-4, IL-5, and IL-8 and mouse IL-4, IL-6, and TNF-alpha ELISPOT assays, allowing one to remove a large number of adhered cells without hindering ELISPOT assay performance. However, Accumax was incompatible with human IFN-gamma, mouse IFN-gamma, canine IFN-gamma, and rat IFN-gamma ELISPOT assays because Accumax reduced the intensity of staining and the number of spots formed.

Simultaneous detection of multiple cytokines in ELISPOT assays.

Living in the era of multiplex detection systems, it appears attractive to develop enzyme-linked immunospot (ELISPOT) assays for the detection of more than one cytokine released by the same cell. However, despite technical simplicity in building such an assay, several factors have to be considered when designing multiplex ELISPOT assays. We have used four capture antibodies (hIFN-gamma, hIL-2, hIL-4, and hTNF-alpha) either in combination or individually to coat polyvinylidene difluoride membrane-backed Millipore 96-well plates. Several cell stimulations were also used, including Concanavalin A, Phorbol Myristate Acetate (PMA) and calcium ionophore (CaI), phytohemagglutinin, CD3e, and lipopolysaccharide. Biotinylated antibodies were used either individually or combined together to detect secreted cytokines. We have found that when plates were coated with all four capture antibodies and captured cytokines were detected using either one detection antibody or all four detection antibodies combined together, fewer spots could be seen when compared with a plate coated with a single capture antibody followed by using its matched detection antibody counterpart. Interestingly, negative interferences between antibodies were less profound when detection antibodies rather than capture antibodies were mixed together.