Several questions regarding your protocol:
1. What concentration is the coating Ab? In my experience, 1 mg/well
gives the best signal:noise ratio.
2. Did you try titrating your coating and detection Abs? I generally
begin by doing a checkerboard of different concentrations of both. I
usually begin my coating Abs or Ags at 0.1 mg/well, and go up to 10
mg/well. Detection Abs I generally try at dilutions of between
1:5000-1:20000.
3. Did you conjugate your detection Ab yourself? Could there be any
free biotin in the Ab prep which might be sticking to your wells?
Jay Mone'
