Sandwich ELISA, Highly Sensitive

Sandwich ELISA is a less common variant of ELISA, but is highly efficient in sample antigen detection. Moreover, many commercial ELISA pair sets are built on this sanwich ELISA.

The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and detection antibody). The antigen to be measured must contain at least two antigenic epitope capable of binding to antibody, since at least two antibodies act in the sandwich. Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in Sandwich ELISA systems.

Monoclonal antibodies recognize a single epitope that allows fine detection and quantification of small differences in antigen. A polyclonal is often used as the capture antibody to pull down as much of the antigen as possible. The advantage of Sandwich ELISA is that the sample does not have to be purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive than direct or indirect ELISA), but lower than ELISpot.

Sandwich ELISA procedures can be difficult to optimize and tested match pair antibodies should be used. This ensures the antibodies are detecting different epitopes on the target protein so they do not interfere with the other antibody binding.

The steps are as follows:

  • 1. Prepare a surface to which a known quantity of capture antibody is bound.
  • 2. Block any nonspecific binding sites on the surface.
  • 3. Apply the antigen-containing sample to the plate.
  • 4. Wash the plate, so that unbound antigen is removed.
  • 5. A specific antibody is added, and binds to antigen (hence the 'sandwich': the Ag is stuck between two antibodies);
  • 6. Apply enzyme-linked secondary antibodies as detection antibodies that also bind specifically to the antibody's Fc region (non-specific).
  • 7. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
  • 8. Apply a chemical that is converted by the enzyme into a color or fluorescent or electrochemical signal.
  • 9. Measure the absorbency or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.

The image at the bottom includes the use of a secondary antibody conjugated to an enzyme, though, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme. However, use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect.

By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay.


Sandwich ELISA Schematic Procedure:

(1) Plate is coated with a capture antibody;

(2) sample is added, and any antigen present binds to capture antibody;

(3) detecting antibody is added, and binds to antigen;

(4) enzyme-linked secondary antibody is added, and binds to detecting antibody;

(5) substrate is added, and is converted by enzyme to detectable form.

Sandwich ELISA advantages :

  • 1. High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected
  • 2. Suitable for complex samples, since the antigen does not require purification prior to measurement
  • 3. Flexibility and sensitivity, since both direct and indirect detection methods can be used

Sandwich ELISA Protocol is shown in a different section.

Sandwich ELISA is a common tool to diagnose Influenza, e.g. H5N1 (Avian Flu) Hemagglutinin ELISA kit. In addition, a description of the application of sandwich ELISA to home pregnancy test can be found here.