High background ELISPOT

Discover solutions for high background in ELISPOT

How can I resolve issues relating to an 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.

What do I do if my plate has 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.

Why are there 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.

What should I do if my 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.