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.
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?
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?
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.
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?