My protocol of ELISA is
Capture Antibody coating (4 micrograms per ml) incubation overnight.
Blocking (1% BSA in PBS) incubation for 1 hour.
Sample loading (100 microliters of BAL fluid diluted in Blocking buffer) incubation for 2 hours.
Detection Antibody ( 1 microgram per ml) incubation for 1 hour.
HRP: incubation for 30 mins
TMB and Stop after 15 mins.
OD for BAL fluid of naive is about 0.3500
Dear Sabin,
the high background signal may originate from unspecific binding of the detection antibody. The chosen concentration of 1 µg/ml, a 1:1000 dilution form a 1mg/ml stock solution, is rather high and using lower concentrations like 0.1 µg/ml or less may reduce the high background.
Regards,
Martin
Hi Sabin,
You can optimize your protocol by increasing the dilution of your capture and/or detection antibody, as well as increasing the concentration of the blocking reagent.
Good luck,
Grace
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