> 1.. What concentration is the coating Ab? In my experience, 1 mg/well
> gives the best signal:noise ratio.
>
This is probably too much antibody (1mg/mL) to coat an ELISA plate and a
waste of antibody. Most people coat with approximately 4ug/mL of antibody
(which is 200 ng/well if you add 50ul/well).
>> 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.
If you don't know the appropriate parameters for an ELISA, it is a good
idea to titrate both the coat and the conjugated antibodies. I would
perform 4 fold dilutions above and below the normal coating (4ug/mL) for
the coat and then for the conjugate I would look at dilutions between
1:800 and 1:10,000.
>
Steph
