In the human IFN-gamma plus kit, it suggests 0.22 um filter for the primary antibody solution, and 0.45 um filter for substrate.  I am guessing the primary filtration would be to remove aggregates? What is the reason for filtering substrate?  would 0.22 work for the substrate as well?  or 0.45 work for the primary antibody filtration step.  thank-you for the information!

Hi Wendy, welcome to the forum!



	When the ELISpot substrate sits in its brown bottle there is a low level of precipitate formed inside of the container over time. This can lead to artefacts if you add the substrate directly to an ELISpot plate. By pushing the substrate through a 0.45um filter, you remove precipitates and you avoid this potential problem. You could in theory also use an 0.22um filter for this process, but it builds up a lot of resistance since the smaller filter quickly gets clogged with precipitate. Its doable, but you have to push like crazy if you try to do 12ml in one go.  



	In order to remove antibody aggregates you have to use 0.22um due to their small size. I believe 0.45 will be too large.

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