Nonspecific staining in ELISpot assay is a major obstacle in accurate quantification of experimental data. The appearance of nonspecific spots may be caused by different factors including cell- and immunoassay-related issues. In our study, we have shown that nonspecific spots can result from either cells or their debris sticking to the membranes in ELISpot plates, as well as by impurities in wash buffers and precipitation of aggregated detection antibodies. Although there is a growing interest in using Fluorospot assays allowing for simultaneous detection of multiple cell-secreted proteins, it appears that these fluorescence assays are more susceptible to developing nonspecific profiles resembling specific spots. In this chapter, we outline necessary ELISpot controls that need to be employed to tell the difference between bona fide spots vs. stained artifacts.
Keywords: ELISpot; Mitogens; Negative control; Nonspecific spots; Positive control.