Protocol for Sandwich ELISA

Materials

• Maxisorb 96-well plate
• 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

1. 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.
2. Block the surface with blocking buffer for 1 h at RT and 700 rpm.
3. Wash the plate three times with washing buffer (at least 5 min per wash).
4. Apply antigen diluted in blocking buffer and incubate for 2 h at RT and 700 rpm.
5. Wash three times with washing buffer.
6. Apply detector antibody diluted in blocking buffer (dilution 1 : 1000 up to 1 : 8000) and incubate for 2 h at RT and 700 rpm.
7. Wash three times with washing buffer.
8. 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.
9. Wash three times with washing buffer.
10. Add 100 µl substrate solution for development.
11. Stop the reaction after 5 - 10 min with 100 µl stop solution and read the absorbance at 450 nm (ref: 620 - 650 nm).