A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. 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 to the right 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.
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