Sandwich ELISA

Sandwich ELISA test protocol

What is an ELISA assay?

ELISA strategies (Sandwich ELISA/ capture, direct or indirect assay) are in simple terms any wet lab analytic biochemical assay.  These assays make use of solid-phase EIA (enzyme immunoassay) to detect antigens.

Sandwich ELISA - types of tests

ELISA history in a nutshell:

1960:  Roslyn Sussman Yalow and Solomon Berson describe radioimmunoassay in a paper. Radioimmunoassays use radioactive labelled antigens or antibodies to conduct immunoassays. An alternative method is, however, required due to the potential health threat caused by radioactivity.

1966:  Jerker Porath publish a paper describing the following protocol:

Horseradish peroxidase (or similar enzymes) reacts with ABTS or TMB to produce a colour.  This colour is used as a signal.  However, this particular signal has to be associated and linked to an antibody or an antigen in a particular sample.  In this protocol, the antibody is fixed to a container surface and the excess antibody or antigens are removed with a washing technique.  I.e. the immunoassay requires preparation.

1971:  Dutch and Swedish scientists publish papers describing a protocol which is known today as Enzyme-Linked Immunoassay or enzyme assay.

2012: Scientists manage to obtain a colour (visible to the naked eye) with the following protocol:

The scientists use nanoparticles as a chromogenic reporter in an extremely sensitive enzyme-based ELISA test.  This indicates detection using attogrammes (10−18 grams) analyte.  In this test, red indicates negative, and blue indicates positive results.  At this point, the test only confirms the presence/absence of an analyte.

Sandwich ELISA capture assay

ELISA capture assay is also known as the Sandwich assay:
This particular assay is the most powerful and highly sensitive (2-5 times more sensitive than direct or indirect) assay to detect sample antigen.  Sandwich ELISA uses a capture and detection antibody. The antigen which is measured requires at least two epitopes.  Sandwich ELISA systems use either monoclonal or polyclonal antibodies:

  • Polyclonal antibodies: capture antibody in order to pull down as much as possible antigen.

  • Monoclonal antibodies for quantification and fine detection of small differences in antigen.

Sandwich ELISA test

One of the biggest advantages of Sandwich ELISA is that this technique does not require sample purification prior to assay.

Sandwich ELISA protocol

Note:  Enzyme substrates may be carcinogenic.  It is also considered hazardous material.  Please follow safety instructions when handling the substrates.

  1. Purify both antibody preparations and label at least one.
  2. Consult manufacturer guidelines to find the best protein binding type of plate.  In general, and for most assays, a PVC microtiter place is best.
  3. Add unlabelled antibody solution to each well – i.e. bind unlabelled antigen. The type of assay will determine the volume. At least 1 μg/well ensures maximal binding. This might be higher than well capacity, but it ensures rapid binding. Save and re-use excess binding solution.
  4. The plate has to incubate overnight at 4 °C. This will allow for complete binding.
  5. Rinse the plate twice with PBS. Use a squirt bottle for this process, as it is easy to use. Flick the plate. This removes the antibody solution.PBS.
  6. Use a blocking buffer to saturate the remaining protein binding sites on the plate.
  7. Incubation: two hours or overnight at room temperature – preferably in a humid atmosphere.
  8. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer.
  9. Fill the wells to the top with 3% BSA/PBS with 0.02% Sodium Azide. Incubate for 2 h to overnight in a humid atmosphere at room temperature. Note: Use Sodium Azide or horseradish peroxidase as an inhibitor. Sodium Azide must NOT be included in washing solutions or buffers if HRP-labelled antibodies are used for detection.
  10. Rinse plate/wells with PBS 2 X.
  11. Titrate 50 μL antigen solution to the plate. All dilutions must be done in a blocking buffer.
  12. Incubation: minimum 2 h in a humid atmosphere at room temperature.
  13. Wash or rinse the plate with PBS 4 X.
  14. Conduct preliminary experiments to determine the amount of labelled second antibody to add. Use excess second antibody and do all dilutions in blocking buffer.
  15. Incubation: two hours or more in a humid atmosphere at room temperature.
  16. Rinse/wash the plate several times. Change the PBS with every rinse.
  17. Follow manufacturer instructions to add substrate. Incubate according to instructions.
  18. Use an ELISA plate reader to measure the optical densities at target wavelengths.


Use Sandwich ELISA assays when:
– You have unprocessed or not refined samples to quantify antigens.
– You need a highly sensitive method to test raw samples.

Sandwich ELISA uses 2 antibodies to detect (fixed on a microplate) and capture antibodies that capture the antigen between the two antibodies, the same way an edible sandwich captures cheese and tomato.

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About Marinda Stuiver

Dip, NHDip Microbiology (Vet) TUT (1989)

Marinda Stuiver is a qualified Veterinary Microbiologist with more than 12 years of laboratory experience.

Her experience includes histology, Electron Microscopy, and ELISA testing. Following the sale of the laboratory where she worked, she ventured into the commercial side of the business.

Marinda sold IDEXX ELISA test kits to various laboratories in Southern Africa (Zimbabwe, Botswana, Malawi) and trained laboratory staff to set up and conduct testing.

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Illustrations on this page courtesy FAVPNG