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High background ELISPOT

Discover solutions for high background in ELISPOT

Over-developed plate

Possible causes

Solution

  • Reduce developing time.

  • Only incubate for 1 hour with the enzyme substrate. Exceeding 1 hour incubation with the enzyme substrate may result in increased background color.

Plate not dried properly

Possible causes

Solution

  • Dry the plate longer before reading, keeping it protected from light. Drying overnight at 4oC may help increase the contrast between background and spots.

Too many cells secreting cytokine/protein of interest

Possible causes

Solution

  • Reduce the number of cells per well. This will require optimization. You may also need to optimize the concentration of stimulant used.

Wash steps not sufficient

Possible causes

Solution

  • Wash both sides of the membrane with distilled water before and after color development. Some reagents may leak through the membrane into the base of the plate, and these can cause high background if not washed away.