• 21. Add 100 µL of developing buffer to all wells using a multichannel pipette and incubate at room temperature for at least 2 h. 
  
  

• 22. Wash the ELISPOT plate: 
  

• i. Twice with 200 µL per well of PBS-T buffer, pouring off the wash buffer between each wash step. 
  
  

• ii. Twice with PBS-T buffer using 200 µL per well and the VPW. 
  
  

• iii. Three times with 200 µL per well of 1X PBS, using the VPW. 
  
  

• iv. Once with 200 µL per well of filtered H₂O. 
  

• 23. Add 100 µL per well of Vector Blue solution and incubate for ~5 min at room temperature. 
  
  

• 24. Remove Vector Blue solution and wash the ELISPOT plates: 
  

• i. Three times with 200 µL per well of 1X PBS, pouring off the wash buffer between each wash step. 
  
  

• ii. Three times with 200 µL of 1X PBS, using the VPW. 
  

• 25. Activate AEC buffer by adding 4.8 µL of H₂O₂ to 10 mL of AEC buffer and filter through a 0.45-µm filter. Add 100 µL per well of activated AEC buffer to each well and incubate up to 5-10 min. 
  
  

• 26. Wash the ELISPOT plate: 
  

• i. Twice with 200 µL per well of filtered H₂O, using the VPW. 
  
  

• ii. One minute under running tap H₂O. Remove the bottom of theELISPOT plate and wash both sides of the membrane. 
  

• 27. Air-dry the plates. 
  
  

• 28. Take a picture of the wells and count spots using the CTL-Immunospot Analyzer as described by the manufacturer. If a picture of the plates cannot be taken and counted on the same day, keep theplates away from light until it can be done. 
  For counting spots, sensitivity and compensation are set using wells coated with either anti-IL-2 or anti-IFN- coating buffer (Step 3), in which cells were stimulated with one of the positive control stimuli,i.e., CEF or anti-CD3 mAb (Step 13). Wells coated with anti-IL-2coating buffer should contain only blue spots. Wells coated with anti-IFN- coating buffer should contain only red spots. 
  

• i. Set sensitivity for each color separately, such that a maximumnumber of spots are detected in each of the anti-IL-2 or anti-IFN- coated wells. 
  
  

• ii. Using the software’s embedded algorithm, set compensation such that no blue spots are detected in the wells coated with anti-IFN- coating buffer and that no red spots are detected in the wells coated with anti-IL-2 coating buffer. 
  Using these settings, a spot that represents the footprint of both IFN- and IL-2 secretion appears purple and is detected when the color of a spot meets the threshold at the intersection of both single colors. 
  
  

• iii. Count the cells in each of the three types of antigen-specific cell populations: single IFN- responding cells, single IL-2 responding cells,and IFN-/IL-2 dual cytokine responding cells. 
  
  

• iv. Always audit the counts obtained from the software well-by-well. 
  See Troubleshooting. 
  

TROUBLESHOOTING

Problem: Wells leak coating buffer.

[Step 10]

Solution: Treatment with MeOH in Step 1 may be too long. Make sure exposure of the wells to MeOH is kept to a minimum. If the problem persists, try less concentrated MeOH solutions or replace MeOH with ethanol.

Problem: The positive control is weak or negative.

[Step 28]

Solution: Consider the following:

• 1. Avoid using cells for which viability is <70% after thawing (Step 8).
  
  

• 2. Avoid cell stress, such as prolonged exposure to concentrateddimethyl sulfoxide (DMSO), insufficient medium, insufficient CO₂, or prolonged exposure to cold temperatures. 
  
  

• 3. Increase incubation time in Step 23 and/or Step 25. 
  

Problem: Spot quality is poor.

[Step 28]

Solution: Spot quality can be affected in several ways:

• 1. A spot with a white center is the result of improper washing of the plate during Step 17; cells remain bound to the membrane and create a "ghost" image during development. Make sure plates are washed vigorously. 
  
  

• 2. Double spots or spots with a cobweb appearance result from plate displacement occurring during Step 16. Ensure plates are not moved during incubation and that the incubator is not located in an active area or near vibrating equipment (e.g., a centrifuge). 
  
  

• 3. Spots that are too dark with a black center make compensation with the counting instrument difficult. Reduce incubation time in Step 23 and/or Step 25. 
  

Problem: The response to background stimuli is elevated.

[Step 28]

Solution: Consider the following:

• 1. Avoid cell stress (see solution above for the positive control being weak or negative). 
  
  

• 2. Put cells on a shaker during Step 9 to avoid cell clumping. 
  
  

• 3. In wash steps using R10 and R20 media, screen lots of fetal bovine serum (FBS) to identify ones supporting low background. Some lots result in increased background and it is recommended that these be tested prior to use. If the problem persists, replace FBS with human AB serum. 
  
  

• 4. Check reagents for contaminants. 
  
  

• 5. Filtration may remove precipitated material that could causebackground. 
  

DISCUSSION

Immune monitoring methods have become essential tools for understanding the correlates of protective immune responses. The dual-color ELISPOT assay described in this protocol allows for the simultaneous detection of antigen-specific T cells secreting IFN- and/or IL-2. The rationale for selecting cytokines to target in a dual-color ELISPOT assay may vary based on the experimental question being posed. IFN- secretion is an antiviral T cell function that isresistant to functional exhaustion in the course of infectionscharacterized by high persistent antigen load and it is the cytokine most frequently assessed in standard ELISPOT assays used to monitor vaccine trials (Shiver et al. 2002; Wherry et al. 2003; Harari et al. 2005; Hill et al. 2007; Kester et al. 2008). IL-2 secretion, on the other hand, is sensitive to functional exhaustion in a setting of high persistent antigen load. However, it tends to identify responses that are more likely to be multifunctional, i.e., have the ability to proliferate and effectively control viral replication (Wherry et al. 2003; Harari et al. 2004; Zimmerli et al. 2005).

Intracellular cytokine staining (ICS) is also used to characterizeantigen-specific immune responses. It has the advantage of allowingfor extensive phenotyping and functional analyses of the respondingcells. However, ICS usually requires a greater number of PBMCs per condition tested, and it is more expensive and less amenable to high-throughput screening than the ELISPOT assay. The ELISPOT assay is at least as sensitive (if not more so) than ICS for detecting antigen-specific cells (Sun et al. 2003; Streek et al. 2009). In resource-limited settings, comprehensive screening with the dual-color ELISPOT could be used to identify specificities that could later be further phenotypically and functionally characterized by multiparametric flow cytometry.

Although both CD4+ and CD8+ T cell responses can be detected with the dual-color ELISPOT, the use of optimal peptides or overlapping 15-mers usually favors the detection of antigen-specific CD8+ T cells (Boulet et al. 2007; Ndongala et al. 2009). However, the ELISPOT assay also allows for the stimulation of T cells with complex antigens such as whole proteins, cell lysates, or whole or apoptotic cells. In the case of complex antigens, these would need to be pre-incubated with antigen-presenting cells (APC) (Schmittel et al. 2000). In this case, antigens would be processed by the APC and appropriately presented to CD4+ and CD8+ T cells, with co-stimulatory signals, according to each cell’s specific requirements. In this context, CD4+ T cells may be more effectively stimulated and, therefore, their responses more accurately detected. In addition, stimulation of T cells with APCs previously exposed to the antigen may favor the detection of antigen-specific cells that were not previously primed in vivo. If the dual-color ELISPOT protocol is adapted to utilize antigen-pulsed APCs as a stimulus, then the APC-priming procedure and incubation time with the responder cells should be optimized.

Another caveat of this method is the decreased magnitude of IFN- responses, by ~15%, in wells where both IL-2 and IFN- are captured when compared to wells coated with IFN- alone (Boulet et al. 2007). This may be a consequence of the mAb-mediated sequestration of IL-2, which is a known inducer of IFN-. Depending on the type of study undertaken, it is therefore necessary to determine whether the information gained from simultaneously detecting both cytokines outweighs the cost of a decreased IFN- magnitude.

The dual-color ELISPOT assay has been used to demonstrate theimportance of the link between the predominance of different T cell functional subsets in HIV-infected individuals with different clinical outcomes or at different stages of disease (Peretz et al. 2007; Ndongala et al. 2009).