Following stimulation, cells are removed, wells extensively washed, and a second analyte-specific Ab is applied. At this and all subsequent steps, washing is critical for the complete removal of cells, nonspecifically bound Ab, and detection reagent. Incomplete removal of unbound reagents will lead to an overall increase in background signal (see troubleshooting chart).
Detection - Chromogenic vs. Fluorescent Options
ELISpot assays may be performed either with antibodies directly conjugated to the detection motif (enzyme or fluorochrome) or as a two-step process involving a biotin/streptavidin-conjugated Ab pair.
While the two-step process offers greater intensity due to signal amplification, and therefore may be preferable in cases where cytokine production per cell is low (allergy/Th2 responses), this protocol could create a greater potential for background staining due to nonspecific interaction with the coating Ab. With enzymes, such as horseradish peroxidase (HRP), a precipitating substrate (TMB or AEC) is used for spot detection. Due to HRP’s high turnover rate, spot development is fast (≤5 minutes).
By contrast, spot development using alkaline phosphatase-conjugated Abs is far slower but with appreciably lower background. For chromogenic assays performed on MultiScreen®HTS plates (those with underdrains), it is recommended that the underdrain be removed prior to substrate addition; failure to do so can result in high background staining. Once removed, plates should be propped up to minimize membrane contact. To enhance spot visualization, plates should be dried without a lid, upside down, at room temperature for several hours. For long-term storage, plates should be kept in a dark, dry place at room temperature to prevent bleaching of spots.
As previously discussed, the use of fluorescent conjugates offers significant advantages over colorimetric schemes especially for dual cytokine applications or where greater quantitative assessments of individual spots is desired. While FITC- and Cy3-conjugated Abs are commonly used, the choice of fluorescent probe is limited only by the availability of conjugates and detection platforms.