Problems with IFNy ELISpot

Hi

I have been using the ELISpot assay and recently I have developed a few problems. I was wondering if anyone had had similar problems and could advise me.

Until now everything had been working very well. To start with I noticed excess spots in the 'media only' wells and now I appear to be getting very little/no spots whatsoever in some plates.

I use PMA and Ionomycin as a control for each sample and i'm looking for responses against PPD. We use standards for each plate and these have all been working fine so we know it isn't a method problem.

I have changed the plates and media. I use Mabtech antibodies. I do not think it is the antigens as on some occasions when using the assay on multiple plates at the same time, some have produced nice spots and others have produced none (apart from the standards)

Any advice greatfully recieved.

Lottie