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
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 |
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| 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 |
|
| Inadequate color development |
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| 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 |
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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 |
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