Using human serum as first antibody in Western/ELISA - (Jun/28/2012 )
Because we could not locate the specific antibody against our HAV epitope, we use HAV vaccinated patient serum as first antibody in Western and indirect ELISA. However the background of negative control, which only applying serum(first Ab)+2nd Ab (anti-human-AP conjugated) was too strong. Tried different blocking with 5%Milk, 3% BSA, and 0.5%TBST, and did not work. After blocking also tried to block with horse serum or FBS, and both did not work. Anyone has any suggestion? Your suggestion will be appreciated.
Titrate the serum - start at the concentration you are currently using and dilute down to as low as 1:10,000 in blocking solution. Run the same samples (+ve and -ve) on a gel separating each group with a ladder so that you have a number of strips of membrane with the same samples on them that can then be detected using the different dilutions.
Thanks. Can I try out the conditions with Dot blotting? It seems to be faster.
Sure, but be aware that if you have non-specific binding (other bands on a western membrane) from the serum, you won't be able to tell if the dot-blot is accurate for the dilution as any binding will show signal.