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False positives in ELISPOT

Find solutions for false positives in ELISPOT.

Check for false positives by running a media negative control.

Cell culture contamination (dust and microbial)

Possible causes

Solution

Dust or microbes have entered your sample.

  • Keep reagents as sterile and clean as possible.

  • Ensure your cell culture technique is aseptic.

  • Reagents can be filtered using a low protein binding syringe 0.2 µm pore size.

Cells still on the membrane, cell debris

Possible causes

Solution

Cells left on the membrane will give irregular-shaped spots.

  • Ensure all the cells are washed from the membrane with PBS Tween 20 before secondary antibody incubation.

Contaminating platelets (when using PBMC prepared from blood samples)

Possible causes

Solution

PBMC preparation needs to be efficient to prevent contamination.

  • PBMC preparation needs to be efficient. Wash the plate well after cell culture stage.

Mitogens and other factors in the serum are stimulating the cells

Possible causes

Solution

Mitogens and other factors in the serum may be stimulating the cells.

  • Heat inactivate the serum.

  • Also see “Poorly defined spots” regarding plate movement

Secondary antibody aggregates

Possible causes

Solution

Secondary antibodies have aggregated.

Any secondaries used in your experiment can aggregate if stored improperly.

  • Filter the secondary antibody.