ELISpot Troubleshooting

The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the enzyme-linked immunospot (ELISpot) assays. ELISpot Protocol is also available for you. For further assistance, please contact us.

Contents

High Background

No Spots/Very Few Spots

Inconsistency in Results Between Wells

High Background

Possible Cause	Recommended Solution
Inadequate wash steps	Wash both sides of the membrane carefully. Increase the washing times.
Too many cells secreting cytokine/protein of interest	Optimize the number of cells per well. Optimize the concentration of stimulant used.
Plate not dried properly	Dry the plate longer before reading.
Over-developed plate	Reduce developing time.

No Spots/Very Few Spots

Possible Cause	Recommended Solution
Not enough cells secreting cytokine/protein of interest	Optimize the number of cells per well.
Ensure the cells are stimulated correctly	Positive stimulation control is necessary.
Cells not incubated for long enough or may take time to respond to stimulant	Increase the cell incubation time. Use indirect method (pre-treat cells with stimulant).
Inadequate color development	Monitor color development with an overhead microscope. Ensure that the developing reagents have been stored correctly and have not lost activity.
Not enough primary or secondary antibody	Optimize the concentration of the primary and/or secondary antibody.

Confluent Spots

Possible Cause	Recommended Solution
Too much antibody	Reduce primary antibody concentration.
Prolonged cell culture	Reduce cell culture step incubation time (not to exceed 24 h).
Cells over-stimulated	Reduce the amount of stimulant in the culture media. Culture for a shorter amount of time.

Poorly Defined Spots

Possible Cause	Recommended Solution
Membrane not pre-treated	Ensure membrane is adequately pretreated with 35% ethanol. Wash well with PBS three times afterwards.
Plate movement during cell incubation	Do not allow the plate to move during cell incubation.
Coating antibody not concentrated enough	Increase coating antibody concentration.
White spots in the middle of a normal spot	This means the enzymatic conjugate has run out of substrate. Therefore, a higher concentration of second antibody and substrate is required.

Blank Areas

Possible Cause	Recommended Solution
Membrane not pre-treated	Ensure membrane is adequately pretreated with 35% ethanol. Wash well with PBS three times afterwards.
Membrane not washed adequately after ethanol treatment	Wash the membrane thoroughly.
Membrane has dried out at some stage	Ensure membrane does not dry.
Cells unevenly distributed	Mix cells gently to obtain a homogenous suspension before pipetting the cells into the wells.
Pipette tip touched the membrane	Take care with pipetting steps, particularly with washing.
Formation of foam	During washing, foam formation can occur. Squirt bottles with narrow spouts produce excessive foam, preventing an effective and uniform wash.

Blank Center

Possible Cause	Recommended Solution
Damage from washing	A gentler washing procedure is required.

False Positives

Possible Cause	Recommended Solution
Secondary antibody aggregates	Filter the secondary antibody or use fresh one.
Cells still on the membrane, cell debris	Cells left on the membrane will give irregular shaped spots. Ensure all the cells are washed from the membrane.
Contaminating platelets when using peripheral blood mononuclear cells (PBMCs) isolated from blood samples	PBMC preparation needs to be efficient. Wash the plate well after cell culture stage.
Cell culture contamination	Keep reagents as sterile and clean as possible.
Mitogens and other factors in the serum are stimulating the cells	Heat inactivate the serum.

Inconsistency in Results Between Wells

Possible Cause	Recommended Solution
Edging effect within the plate	Ensure a well-mixed single cell suspension of sample is used when adding cells to the wells. Do not stack the plates during cells incubation.