Hi all,
I have been doing immuno-PCR on urine antigen detection, however I am frequently getting contamination/non-specific binding I am not sure.
The procedure is combining ELISA and PCR together to boost sensitivity for antigen detection. It is usually antigen coating (in this case we use healthy control urine for negative controls and antigen spiked urine for positive controls) on a 96-well plate, followed by a blocking stage and then primary and secondary antibody coating and coating with biotinylated DNA which will bind to the secondary antibody. Then add master mix and primers for PCR running, then run the agarose gel.
I have got so frequent bands in negative controls, I have tried use new set of primers/master mix (even the only master mix primer control is showing band but I have used new opened master mix and newly-prepared primers), tried use new 96-well plate, sanitise the pipettes and always use filtered tips; changing the percentage of blocking buffer (now using 4% BSA). But despite this, the bands are still showing in negative controls (but not for all negative controls sometimes).
in addition to this, I also noticed the problem of non-specific binding of using Biotinylated DNA-Stretavidin (I suspect). when I don't add this complex and the negative control will stay as negative. But 8/10 times I will get non-specific binding in negative controls/negative samples (no antigen). I have changed wash buffer, adjust blocking agents and everything I could try. But still getting non-specific bindings.
Really appreciate your help about this, Thanks!!
I would move to another researchers area (in case yours is contaminated) and set up using their pipettes and plasticware and as many of their pcr reagents as possible. Run assays with 5 NTCs and one positive control only. If this works clean then introduce one feature at a time until the contaminations shows up. While doing this strip your pipettes down completely and clean them inside and out. Often pcr product gets into the barrel of the pipette and dries there and this dried material can fall into the assay reagents or tubes. I think that dust in a contaminated working area ( from pening tubes/aerosols or spillage of electrophoresis buffer (used) and internal pipette barrel contamination are the most common reasons for contamination
- Badges
- Science topic