Here are some more details:
- We use a commersial kit for ELISA
- Even though the samples are thoroughly diluted, some of the wells give
more absorbance then they should, and some give less then they should.
when plotting the results we can see a curve, but it has lots of peaks and
valleys which shouldn't be there.
- We have tested the protocol with different washing procedures (fewer
steps, less tween-20) but no result.
- We've tried another protocol from a completely different ELISA-kit (for a
different protein) with slight adjustements (off course we did not change
the antibodies) but with no success.
- It can not be the plates we're using.
Have anyone here experienced similar strange results? I understand that it
would have helped if I could name the specific cytokine, but I can't. I
hope that someone have seen anything like it and could direct us to the
cause of the problems in such a way that our troubleshooting would be
One more thing, we've experinced that shaking during incubation periods
give greater results and also less standard deviation for the curves
obtained when performing ELISA. This was not mentioned in the protocols
we've received with the kits. This may be a general hint to anyone out
Trond Erik Vee Aune wrote:
>> I'm having trouble with my protocol for sandwich ELISA of a cytokine. I
> get almost random results with more result from wells downstream in a
> dilution series than upstream. What can be wrong?
>> Trond Erik