I’m wondering if you can help with this issue. Currently working on In-house ELISA using Nunc thermo scientific plates and Gene script spike protein it started to give too much signal. I thought it was contamination so used new and freshly prepared materials, change the coat protein to sinobiological , washing process from automated to manual. followed all the recommend process to solve this issue i.e sufficient amount of tween , washing etc. but, it still same issue.
Is there something I missed? Any suggestions is welcome
Hello Hind Albalawi,
could be several things. Which plates are you using? Switching from MaxiSorp to PolySorp could be a Thing for you. Personally i would recommend staying with automated washing. Also adjusting Casein-concentration in sample buffer might be it. Which buffer you are using for blocking? And maybe changing your conjugate-concentration could help you.
Could be a problem at any step but what did you use for blocking? Did you do the blocking step properly and used blocking buffer to add your samples etc? Secondly, after the addition of Avidin HRP or Biotin HRP, the washing step is extended generally to 5-6 washes and you can leave the wash buffer for like a minute before throwing to avoid getting over signal. Also you can decrease the incubation time of HRP etc accordingly and increase the dilution of the antibodies you are using.
Hi Dennis Niewiadomski & Shivani Yadav , Thank you so much for your suggestions,
I run ELISA in the past (following the exact protocol) and worked fine.
It's just the last week as soon as put in the substrate the whole plate turn yellow within few minutes. I can't figure out what went wrong despite that, I used new and fresh materials.
(The blocker is Milk powder in PBS/T and used pNPP substrate)
Hi Hind Albalawi ,
was there any Change of Lots in the materials? Also had once the Problem, that the manufacturer of These had a Change, Maybe something is wrong there.
Best Regards, Dennis
Hi,
You can use 2% FBS for blocking, milk gives a lot of background sometimes.
I Hope, your problem is resolved already.
Good wishes :)
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