ELISpot plates / Pretreatment

 

The use of PVDF membrane plates enables efficient binding of high amounts of capture antibody, provided that the PVDF membrane is treated with ethanol (EtOH) prior to coating. Empirically, different types of plates have been found to react differently to the EtOH treatment and it is critical to use an optimized protocol (see recommendations below).  

 

The EtOH treatment is followed by washing with sterile water before addition of capture antibody. After a correct EtOH treatment, the spots obtained will be of significantly better quality and spot numbers may also often be higher compared to what is seen in non-EtOH treated plates. Noteworthy, treatment with larger volumes of EtOH or longer treatment times than recommended can instead reduce the performance of the assay.

 

MSIP No EtOH	MSIP 15µl/35%/1 min	ELIIP 50µl/70%/2 min	MSIP Precoated
Influence of ethanol (EtOH) treatment prior to the coating step when using PVDF plates. Shown above are: MSIP plate not treated with EtOH; MSIP plate treated with 15µl of 35% EtOH for 1 min; and ELIIP plate treated with 50 µl of 70% EtOH for 2 min. A precoated MSIP plate included for example in the ELISpotPRO kit is shown for comparison. Human PBMC were stimulated with CEF peptide pool and assessed for IFN-γ production (250 000 cells/well).

 

 

Precipitating substrates

 

For the final detection of spots a precipitating substrate is used. This will vary dependent on what enzyme (ALP or HRP) is used in the previous step. As different substrates do not only give different colors (see below) but may also yield different sensitivities the choice of substrate is not trivial. We normally recommend BCIP/NBT for ALP and TMB for HRP as both show excellent sensitivity and give distinct spots that are easy to evaluate.

 

 

ALP                                                                HRP
BCIP/NBT	Fast Red	TMB	AEC
Common substrates in ELISpot. Different enzymes (ALP or HRP) in the ELISpot can be combined with different substrates. Here this is illustrated by using IFN-γ ELISpot with substrates BCIP/NBT and Fast Red for alkaline phosphatase (ALP), and with TMB and AEC for horseradish peroxidase (HRP). Please note that the results were generated in different experiments and therefore the number of spots should not be compared.