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Help needed! ELISA reproducibility issues?


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2 replies to this topic

#1 Ayeshia Beavers

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Posted 25 January 2013 - 08:08 AM

I really need help! I am new to running ELISA plates and I have run them multiple times for the past few weeks but I am still not getting the results I need. I am noticing variations between my duplicates. Recently I had my supervisor run a plate along side me and he had issues with his plate in the same places I had issues. He did point out a pipetting error on my behalf but other than that he said I did everything right, he also checked all of my calculations which were also correct. Does anyone have any suggestions about what could be going wrong, I just find it alarming that he is so experienced with running ELISA plates and his had issues too. :-(

#2 Sotirios

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Posted 28 January 2013 - 12:37 AM

1) Check the coating step. Maybe there is edge effect during this step and the problem is in plate, about reproducibility.
2) Check the room humidity. Many times there is a problem with the humidity existance that cause issues when there is delay during the TMB loading step.
Athanasiou Sotirios

#3 Immuno1girl

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Posted 28 January 2013 - 08:24 AM

It might be your wash buffer (or more likely container) is contaminated. If using an immunowash, clean, replace tubing, and autoclave (if possible). If using a wash bottle, clean and dry before re-using. Wash buffer contamination is the number one cause of erroneous signal in ELISA.




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