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Standard 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, Streptavidin peroxidase (HRP)conjugated

  • Microplate shaker

  • Microplate absorbance reader with filters at 450 nm and a reference wavelength (e.g. 620-650 nm)

Solutions:

  • 0.1 M Na-carbonate, pH 9.6 (store 0.5 M stock at -20°C)

  • Tris-buffered saline (TBS) with 0.05 % Tween 20 (TBST)

  • 5 % skimmed milk in TBST (blocking buffer)

  • Tetramethylbenzidine (TMB) reagent for development

  • 1 M H2SO4 to stop development

Procedure:

  1. Coat 96-well microplate with 100 µl capture antibody (200-400 ng/well) in 0.1 M Na-carbonate, pH 9.6, and incubate overnight at 4°C.

  2. Block the surface with blocking buffer; 1 h at RT and 700 rpm.

  3. Wash plates three times with TBST (at least 5 min per wash).

  4. Apply antigen diluted in blocking buffer; 2 h at RT and 700 rpm.

  5. Wash three times with TBST.

  6. Apply detector antibody diluted in blocking buffer (dilution 1 : 1000 up to 1 : 8000); 2 h at RT and 700 rpm.

  7. Wash three times with TBST.

  8. Incubate with HRP-coupled goat anti-species B antibody or HRP-conjugated streptavidin, diluted in blocking buffer (1 : 5000 - 1 : 10000); 1 h at RT and 700 rpm.

  9. Wash three times with TBST.

  10. Add 100 µl TMB reagent for development.

  11. Stop the reaction after 5-10 min with 100 µl 1 M H2SO4 and read the absorbance at 450 nm (ref: 620-650 nm).







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Synaptic Systems GmbH | Rudolf-Wissell-Str. 28 | 37079 Göttingen | Germany
Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com