ELISpot Optimization

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While ELISpot assays permit frequency determination for very rare events, data interpretation can become ambiguous when:

(1) spot numbers in antigen-containing wells are low
(2) spot counts in negative control wells are elevated
(3) When both spot number is low and background is high simultaneously

Thus, the primary task, even before statistics are employed, must be the optimization of basic assay parameters and reagents to maximize the signal-to-noise ratio. While the use of highly specific ELISpot–validated Ab pairs is key to assay success, proper consideration and execution of a number of other steps are required to ensure optimal performance. Some key considerations from each part of a standard ELISpot assay include: