A sandwich ELISA measures the amount of antigen between two layers of antibodies. One layer is the capture antibody, the other is the detection antibody. The antigen to be measured must contain at least two antigenic sites capable of binding to antibody, with one binding to the capture antibody and the other binding to the detection antibody. Sandwich ELISA is very sensitive (more so than direct or indirect ELISA) and the sample does not have to be purified before analysis.

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Protocol Series:
Sandwich ELISA
Information on the general steps required for carrying out a Sandwich ELISA protocol
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2. A sandwich ELISA measures the amount of antigen between two layers of antibodies. One layer is the capture antibody,
the other is the detection antibody. The antigen to be measured must contain at least two antigenic sites capable of
binding to antibody, with one binding to the capture antibody and the other binding to the detection antibody.
Sandwich ELISA is very sensitive (more so than direct or indirect ELISA) and the sample does not have to be purified
before analysis.
The following slides provide information on the general steps involved. Optimisation may be required.
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3. Sandwich ELISA
1. A surface is prepared to which a known quantity of capture antibody is bound.
2. Any nonspecific binding sites on the surface are blocked.
3. The antigen-containing sample is applied to the plate, and captured by antibody.
4. The plate is washed to remove unbound antigen.
5. A specific antibody is added, and binds to antigen.
6. Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the
antibody's Fc region (non-specific).
7. The plate is washed to remove the unbound antibody-enzyme conjugates.
8. A chemical is added to be converted by the enzyme into a colour or fluorescent or electrochemical signal.
9. The absorbance or fluorescence or electrochemical signal of the plate wells is measured to determine the
presence and quantity of antigen.
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