ELISPOT

[darren at indy.bio.uts.edu.au]

I am trying to optimise an ELISPOT assay for specific B-cells isolated from
rainbow trout to the protein antigen KLH.  When I capture B-cells with a
monoclonal anti-trout Ig Ab I get textbook ELISPOTs from immunised and non-
immunised fish (fewer spots/million cells in the non-immunised fish).  However
when I capture B-cells with KLH coated wells I get large numbers of very small
pin-prick sized spots in the immunised fish, but no spots whatsoever from the
non-immunised fish.  All the papers, books etc I have seen dismiss such small
spots as "artefacts", but don't explain this term or the possible origin of
such spots.  A negative control antigen (BSA) gave absolutely no "artefacts"
or spots.  Can anybody tell me a) what these artefacts are , b) why I am only
getting artefacts but not real spots, and c) suggest a way to overcome this.

Thank you  Darren



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