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Posted 22 June 2017 - 12:55 PM
I have seen similar white spots occasionally before, but I can’t say why they appear with certainty. Since there are normal black spots in the same area, the white spots shouldn’t be due to lack of capture antibodies.
Could it be a solvent-issue? I mean, could the white spots appear due to Tween in the washing buffer or DMSO in the antigen suspension? DMSO might be needed to dissolve a hydrophobic antigen. In our experience, a DMSO concentration of 0.1% is ok, but it shouldn’t be higher than that. If impossible to get down to 0.1%, we at least strongly recommend you should not go above 1% DMSO in your assay – that may harm the PVDF membrane and/or solubilize cell membranes and remains of cell debris that can result in rubbishy membrane appearances. Some antigens might require a higher concentration of DMSO to be dissolved, but once dissolved it is usually possible to dilute the DMSO (to 0.1% for example) without affecting the antigen.
Similar adverse effects apply to Tween, which is a common solvent to add in washing buffers in e.g. ELISA. We don’t recommend Tween for ELISpot; we have first of all seen no advantage of including Tween in the washing steps and you risk introducing adverse effects similar to the ones described above for DMSO. Did you use Tween in your washing buffer?
In any case, as long as the white spots aren’t counted, they shouldn’t interfere with the analysis of the plate and thus the result of the experiment.
My views are in many cases my own and may not necessarily reflect those of Mabtech.
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