[Immunology] ELISA problems ('dead wells')

m.bristow at apu.ac.uk m.bristow at apu.ac.uk
Thu Feb 23 15:01:38 EST 2006


Hi,


I'm experiencing a new (to me) problem with ELISA.  I have an double
sandwich human IgA ELISA using HRP (and TMB as the substrate) using
Greiner Medium bind plates.    The standard curve is good.  I have had
some problems with poor CV% on some runs and we are now seeing 'dead
well' - wells that just don't develop any colour (9 in one 96 well
plate today) and some wells with much more colour than others, giving
us poor CV% in wells.


I can't see how these dead wells could be caused by the overnight
coating of the capture antibody (STAR92) in the fridge or by the
incubation/ shaking stages (can plate shaking cause problems?).  I'm
also confident that this isn't an operator problem.


I'm left with the feeling it is the plate washer (Dynex Ultrawash),
though I can't  think of any way it could be causing it.


I'd appreciate any ideas/ thoughts/guidance on what could be causing
these 'dead wells' and/ or if anybody has had similiar problems to
this.


Thanks,


Matt


Matt Bristow
Stress and Health Research Group
Department of Psychology
Anglia Ruskin University, Cambridge. 


m.bristow at apu.ac.uk



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