Help needed! ELISA reproducibility issues? - (Jan/25/2013 )

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. :-(

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.

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.