Over-Developed Plate with Excessive Background Color (ELISPOT)

Question

Accepted Answer

The entire membrane has excessive background color development, reducing contrast between spots and background. Spots may be obscured by high background signal. Common causes: 1) Enzyme substrate incubation time exceeding 1 hour 2) Plate not dried properly before reading 3) Insufficient washing steps allowing reagent accumulation 4) Foam formation during washing preventing effective and uniform wash coverage Solutions: 1) Limit enzyme substrate incubation to maximum 1 hour; monitor development with microscope 2) Dry plate longer before reading, protected from light; overnight drying at 4°C increases contrast 3) Wash both sides of membrane thoroughly with distilled water before and after development 4) Avoid squirt bottles with narrow spouts that produce excessive foam; use proper wash technique