Validation of ELISpot

[Guest Margot]

Hi,

I have developed an ELISpot protocol that seems to be working quite well. Before I apply the protocol to test more of my precious patient samples, I wanted to check if there are (official) rules for the validation of ELISpot assays in terms of intra and inter-assay variability?

I can see some variability between assays, but I suspect this is because my counting may not be that accurate. I now use tryptan blue staining and manual counting to determine viabilty and cells/mL which might not be ideal as it induces variability. What other methods are recommended?

[Renata]

Hi Margot,

Sorry that we haven’t answered your question until now! Have you already assessed intra and inter-assay variability of your ELISpot assay? In addition to inter- and intra-assay variability, it may also be valuable to assess reproducibility of the assay if more people than just you are going to be performing the experiments.

 

For information on how to assess the precision of your assay and other suggestions for ELISpot validation I would recommend this book chapter by Sylvia Janetzki:

Janetzki S. Important Considerations for ELISpot Validation. Methods Mol Biol. 2024;2768:1-13. doi: 10.1007/978-1-0716-3690-9_1

 

Manual counting can indeed be one of the sources of higher variability. It may be worth investing in a flow cytometry-based cell counter. It’s more accurate than manual counting or image-based automated cell counters and you can exclude both dead and apoptotic cells from your cell count. This way you make sure that you are not overestimating the number of viable cells in your sample. In-house we use Cytek’s Guava Muse Cell Analyzer for cell counting, for example.

All the best,

Renata