The Protein Man's Blog | A Discussion of Protein Research

The Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most efficient laboratory procedures used in detecting the presence and measuring the concentration of a target antigen in a completely unknown sample. Its superior sensitivity and extremely robust nature makes it a great diagnostic tool for medical purposes and is especially useful in identifying potential food allergens and/or testing for certain drugs.

There are several ELISA options, which include the following:

• Direct/indirect ELISA – Direct ELISA uses a labeled primary antibody in analyzing the presence of an antigen while indirect ELISA refers to an ELISA wherein the antigen is bound by the primary antibody and detected by a labeled secondary antibody.
• Competitive ELISA – uses a sample (unlabelled) antigen and an add-in (labeled) antigen to compete for primary antibody binding sites. This procedure is ideally used for crude, impure and complex samples.
• Sandwich ELISA – ideal for quantifying antigens “sandwiched” between the capture antibody (which is immobilized on a surface) and detection antibody. Sandwich ELISA is about 2 to 5 times more sensitive than direct and/or indirect ELISA and offers fast and accurate detection of the antigen in an unknown sample. It is also extremely flexible and can be used for complex samples since the antigen doesn’t need to be purified prior to measurement.

Sandwich ELISA: Getting Down to the Basics

As mentioned earlier, the Sandwich ELISA can be particularly useful in detecting the presence and quantifying the antigen concentration in an unknown sample. Since the protocol uses at least two antibodies, the antigen needs to have at least two non-overlapping antigenic epitopes capable of binding to the antibodies.

The capture and detection antibodies can be monoclonal or polyclonal. Monoclonal antibodies are often used as detection antibodies since they allow for a more accurate detection and quantification while polyclonals are ideally used as capture antibody since they do a great job in binding antigens. For best results, use only match-paired antibodies to make sure the antibodies bind to different epitopes on the target protein and do not interfere with each other’s binding capabilities. 

Sandwich ELISA General Protocol

1. Coat the well of a microtiter plate with capture antibody to render it immobile. To increase assay sensitivity, wash off any unbound antibodies with PBS after incubating overnight at 4^oC
2. Coat the plate wells with blocking buffer. Use 5% non-fat dry milk/PBS to block the remaining protein-binding sites in the coated wells as well as to reduce background and non-specific binding. Incubate for 1 to 2 hours at room temperature or overnight at 4^oC, and wash with PBS.
3. Apply samples. Add diluted samples to each plate. Don’t forget to run samples and standards in duplicates or triplicates, and incubate for 90 minutes at 37^oC to give the target antigens ample time to bind with the immobilized capture antibodies. Remove the samples and rinse the plates twice with PBS to remove any unbound antigens.
4. Apply detection antibody. After adding diluted antibody to each well, cover the plates with an adhesive plastic and incubate at room temperature for 2 hours. Rinse the plate repeatedly (about 4 times) with PBS to ensure that only the antibody-antigen complexes remain.
5. Apply secondary antibody. Add the enzyme-linked secondary antibody (which was diluted in blocking buffer immediately before use) and incubate for an hour or two at room temperature. This will serve as the detection antibody that will specifically bind with the antibody’s Fc region. Wash the plate with PBS to remove all traces of unbound antibody-enzyme conjugates.

Use a chemical substrate to detect signals. Use an appropriate substrate (e.g. HRP, ALP, TMB, OPD, ABTS) to induce a chromogenic, fluorescent or chemiluminescent signal, and measure it using a spectrophotometer or other optical device to determine the presence and quantity of the antigen.

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