Specific antibody from species A as capture antibody
Specific antibody from species B or biotinylated antibody from species A as detector antibody
Goat anti-species B IgG peroxidase (HRP) conjugated or Streptavidin peroxidase (HRP) conjugated
Microplate shaker
Microplate absorbance reader with filters at 450 nm and a reference wavelength (e.g. 620-650 nm)
Reagents
Coating buffer: 0.1 M Na-carbonate, pH 9.6 (store 0.5 M stock at -20°C)
Washing buffer: Tris buffered saline (TBS) with 0.05% Tween 20 (TBST)
Blocking buffer: 5% skimmed milk in TBST
Substrate solution: Tetramethylbenzidine (TMB) reagent for development
Stop solution: 0.25 M H2SO4 to stop development
Procedure
Coat 96-well microplate with 100 µl capture antibody (200-400 ng/well) in coating buffer. Seal the 96-well microplate and incubate overnight at 4°C.
Block the surface with blocking buffer for 1 h at RT and 700 rpm.
Wash the plate three times with washing buffer (at least 5 min per wash).
Apply antigen diluted in blocking buffer and incubate for 2 h at RT and 700 rpm.
Wash three times with washing buffer.
Apply detector antibody diluted in blocking buffer (dilution 1:1000 up to 1:8000) and incubate for 2 h at RT and 700 rpm.
Wash three times with washing buffer.
Incubate with HRP-coupled goat anti-species B antibody or HRP-conjugated streptavidin, diluted in blocking buffer (1:5000 - 1:10000) for 1 h at RT and 700 rpm.
Wash three times with washing buffer.
Add 100 µl substrate solution for development.
Stop the reaction after 5-10 min with 100 µl stop solution and read the absorbance at 450 nm (ref: 620-650 nm).
