A “sandwich” ELISA is used to detect sample antigen. The steps are:
- A surface is prepared to which a known quantity of capture antibody is bound.
- Any nonspecific binding sites on the surface are blocked.
- The antigen-containing sample is applied to the plate, and captured by antibody.
- The plate is washed to remove unbound antigen.
- A specific antibody is added, and binds to antigen (hence the ‘sandwich’: the Ag is stuck between two antibodies). This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.
- Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody’s Fc region (nonspecific).
- The plate is washed to remove the unbound antibody-enzyme conjugates.
- A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal.
- The absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells is measured 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 (which would be direct ELISA). However, the 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. A sandwich ELISA used for research often need validation because of the risk of false positive results.