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Poorly defined spots in ELISPOT

Find solutions for poorly defined spots in ELISPOT.

Coating antibody not concentrated enough

Possible causes

Solution

Coating antibody not concentrated enough

  • For fluorescence – increase the antibody concentrations.

  • Spot quality can help to define if the capture antibody is too diluted or if there is an issue during the coating step.

Inconsistency in results between wells

Possible causes

Solution

  • Do not stack the plates during cell incubation. This will give an edging effect within the plate due to the non-uniformity of temperature distribution.

  • Ensure a well-mixed single-cell suspension of sample is used when adding cells to the wells.

Membrane is not pre-treated

Possible causes

Solution

  • The membrane must be pre-treated with ethanol or the result may be fuzzy, poorly defined spots. It will be difficult for the reader to distinguish these.

Plate movement during cell incubation

Possible causes

Solution

  • Do not allow the plate to move during cell incubation as cells that have moved will create more than one spot. If possible use a dedicated incubator that will not be opened during the incubation. Do not tap the plate after adding cells.

White spots in the middle of a normal spot (more usual with enzymatic detection)

Possible causes

Solution

  • For fluorescence – increase the antibody concentrations.