Troubleshooting Your ELISpot Assay

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Issue 1: High Levels of Background Staining

Were cells washed prior to the incubation step?
Solution or Explanation
Washing prevents the carryover of secreted cytokines in the preincubation medium.

Was the secondary/detection antibody filtered prior to use?
Solution or Explanation
Antibody filtration using Millex® filters (Catalog No.SLHV033RS) reduces background staining or false positive spots that may arise due to protein aggregates.

Was the recommended number of wash steps performed throughout the assay?
Solution or Explanation
As plate washers are less vigorous than manual methods, we recommend 1.5X the standard number of washes if a plate washer is used.

Were sterile technique and reagents employed during assay execution? 
Solution or Explanation
Culture contaminants may result in nonspecific background staining.

Was the membrane dried completely prior to analysis? 
Solution or Explanation
Wet/damp membranes can display a dark blue background color. Drying overnight at 4 °C may increase contrast between background and spots. Drying at temperatures greater than 37 °C may cause membrane cracking.

Was the plate moved/knocked during incubation? 
Solution or Explanation
Due to diffusion, background staining or diffuse spots can arise if plates are moved during the cell incubation step.

Was the percentage of live/dead cells estimated prior to incubation? 
Solution or Explanation
A high number of dead cells may result in high background staining and/or lack of spots.

Was the cell seeding density optimized prior to the commencement of the assay? 
Solution or Explanation
We recommend prior optimization of input cell number and stimuli concentration.

Was the secondary/detection antibody concentration, enzyme conjugate (HRP-Streptavidin or AP-Streptavidin) and enzyme substrate optimized prior to the commencement of the assay? 
Solution or Explanation
Excess biotinylated secondary/detection antibody or enzyme conjugate is likely to contribute to background. A reduction in the concentration of reagents or reaction time will reduce background.

Was PBS filtered prior to use? 
Solution or Explanation
Some PBS formulations may benefit from filtering with a 0.2 μm filter prior to use.


Issue 2: No Spots/Blank Wells

Was a membrane prewetting step performed?
Solution or Explanation
Inadequate pre-wetting may result in an absence of signal, non-staining areas or poorly defined spots due to poor capture Ab binding. Make sure that the 35% EtOH is prepared immediately before use and is a true 35% solution (not 35% of 95% EtOH).

Did the membrane turn gray/translucent after prewetting?
Solution or Explanation
Due to diffusion, background staining or diffuse spots can arise if plates are moved during the cell incubation step.

Have you chosen the correct antibody pairs?
Solution or Explanation
Ensure that the capture and detection Abs react with different antigenic epitopes.

Was the cell seeding density optimized prior to the commencement of the study? 
Solution or Explanation
An absence of spots may indicate that the frequency of responder cells is very low.

Have you stimulated your cytokine/protein of interest appropriately? 
Solution or Explanation
For T cell responses, we recommend using a polyclonal activator such as PHA for a positive control.

Did the culture medium turn yellow during stimulation? 
Solution or Explanation
If so, a high percentage of cells may have undergone apoptotic/necrotic cell death.

Were cells resuspended into a single cell suspension prior to addition to the ELISpot plate? 
Solution or Explanation
Clumping may lead to the underestimation of spot-forming cells and inconsistent results.

Were your cells stored appropriately prior to stimulation? 
Solution or Explanation
Cell viability should be assessed prior to culture set-up and stimulation. We recommend the guava easyCyte™ benchtop flow cytometry system and ViaCount® reagent.

Was PBST (PBS + 0.5% Tween® 20) used for the final wash before spot development? 
Solution or Explanation
Detergents, such as Tween®-20, can inhibit enzyme reactions. Use “PBS only” for final wash steps.


Issue 3: Fuzzy/Poorly Defined/Confluent Spots

Was a prewetting step performed?
Solution or Explanation
Prewetting is not universally applicable to all ELISpots; its requirement is dependent on the inherent hydrophobicity of the capture Ab; therefore, the pre-wetting protocol should be optimized prior to application.

Was primary/capture antibody concentration optimized prior to starting the assay?
Solution or Explanation
A common cause of large, diffuse spots is insufficient capture antibody. It is good practice to determine optimal Ab concentrations before use. Using validated kits will ensure that all reagent concentrations are optimal.

Were plates stacked during the incubation step?
Solution or Explanation
Stacking plates may affect the even distribution of heat across plates or individual wells.

Were the developing reagents allowed to come to room temperature prior to use? 
Solution or Explanation
The HRP and AP enzymatic reaction(s) perform optimally at room temperature. Poorly defined spots may be the result of underdevelopment due to addition of cold substrate.

Was incubation time optimized prior to commencement of the assay? 
Solution or Explanation
The longer cells are incubated, the more cytokine/protein they will secrete, resulting in larger spots that start to merge and become indistinguishable. Incubation time can vary (18-48 hours) according to cell type and cytokine/protein of interest. The amount of stimulant may also require optimization.

Was the plate allowed to dry completely before reading? 
Solution or Explanation
Drying overnight at 4 °C may help increase the contrast between background and spots.