ELISpot Troubleshooting Sessions

#1 & #2
1. Antibody coating
-- Where most problems can arise

Please note!
This step is omitted when using pre-coated plates.
Thus, pre-coated plates solve most problems.
1. Antibody coating
-- Choosing the plate

White MSIPS4W10 plate

Harder to see if you miss adding liquid

to a well.

Clear MSIPS4510 plate

Recommended
These two plates have
“underdrains” that protect the
membrane from fluid splashes

White MAIPSWU10 plate

Higher risk of membrane leakage.

1. Antibody coating
-- Dark blobs formed by liquid splashes in strip plate formats
1. Antibody coating
-- Pre-treat the plate with ethanol

Ethanol makes the membrane hydrophilic and thus allows for a higher
concentration of capture antibodies  spots of higher quality.

EtOH No EtOH

The PVDF membrane resembles a sponge Better quality

of spots
 Provides a larger membrane surface
 Increases the amount of capture antibody
that can be bound
1. Antibody coating
-- Recommended concentration of the capture antibody

For most analytes: 15 μg/mL, 100 μl

To summarize:
• Large surface area (PVDF)
• Increased absorbing capacity (ethanol pre-wetting)
• Recommended concentration of antibody

=
 Optimal amount of capture antibody
 More efficient capture of analyte
 More distinct appearance of spots
 Improved spot count
2. Cell incubation
-- Cell viability is of the essence!
2. Cell incubation

ELISpot works for any cells

• Mouse spleen cells
• Cell cultures
• Cells from mucosal tissue No EDTA!
• Heterogeneous cell populations Choose a heparin- or citrate-based anticoagulant
• Purified homogeneous cells
• Cell clones
• Most common: Human peripheral blood mononuclear cells (PBMCs)

• We prepare PBMCs from blood by Ficoll separation

2. Cell incubation
-- Choose a cell culture medium appropriate for your cells

Q: Can serum-free medium be used in ELISpot?

A: Yes, serum-free medium (containing defined proteins as a

substitute for total serum) can be used, but it should be evaluated
before use.

The same medium should be used throughout the protocol,

including for step B2 blocking of unspecific binding.

RPMI-1640 + 10% FCS

+ HEPES + PenStrep.

Janetzki et al 2010
2. Cell incubation
-- Avoid touching the membrane!
2. Cell incubation
-- Titrate the number of cells per well for each sample type, project, and analyte

Unstimulated Anti-CD3 PHA

25k cell/well

50k cell/well

100k cell/well

200k cell/well

400k cell/well
2. Cell incubation
-- Titrate the number of cells per well for each sample type, project, and analyte

25k cells/well 50k cells/well 100k cells/well 200k cells/well 400k cells/well

Anti-CD3

Unstimulated
3. Analyte capture
3.5 Cell removal
-- No need to have detergents when removing cells

• We do not recommend using detergents like Tween 20 with Mabtech’s ELISpot products
• Detergents can lead to membrane leakage in the wells
• The spots are a few magnitudes larger than the cells, so there is no need to worry that residue
cell debris on the membrane will interfere with spot detection.