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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

Confluent Spots

Poorly Defined Spots

Blank Areas

Blank Center

False Positives

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
  1. Increase the cell incubation time.
  2. Use indirect method (pre-treat cells with stimulant).
Inadequate color development
  1. Monitor color development with an overhead microscope.
  2. 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
  1. Reduce the amount of stimulant in the culture media.
  2. 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 PBMC 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
  1. Ensure a well-mixed single cell suspension of sample is used when adding cells to the wells.
  2. Do not stack the plates during cells incubation.