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Protocols > Standard protocol for sandwich ELISA
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)
Solutions needed
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).