A couple of suggestions, firstly I have used peroxidase to develop an ELISA
and I found that a higher dilution of Ab/enzyme conjugate (maybe 1:10000 -
try a few concentrations its been a while since I did it!) helped. The
second thing I would suggest is using 3% BSA in PBS to block your plates and
using 0.2% BSA in PBS to dilute your standard curve, secondary antibody and
enzyme conjugate. You may want to decrease the length of time your secondary
AB and enzyme conjugate incubations are left (I use 45mins room temp and 30
mins room temp respectively) although this may depend on the antibodies that
you are using. Finally I would definetly recommend that you wash your plate
6-8 times after the enzyme conjugate incubation, this is the most important
washing step.
Good luck!
Andrew
