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Sandwich enzyme-linked immunosorbent assays (ELISAs) are the most frequently used ELISA format. It requires the use of matched antibody pairs, whereby each antibody is specific for different non-overlapping epitopes on the antigen. Firstly, one known as the capture antibody is coated onto the surface of a 96-well plate to facilitate the immobilization of the target antigen. Then the detection antibody binds to the capture antibody-antigen complex. Finally, an enzyme-labeled secondary antibody conjugate specific for the detection antibody is added. In direct sandwich ELISA format, only two specific antibodies are used to sandwich the antigen. One is a capture antibody and detection antibody is another. Either monoclonal or affinity-purified polyclonal antibodies can be used as capture and detection antibodies, depending on cost, the dynamic range, and the sensitivity of the final assay.

The procedure for a sandwich ELISA first requires the well of an ELISA plate to be coated with a capture antibody. The analyte or sample is then added, followed by the detection antibody. According to the detection principle and whether to use the enzyme-labeled capture antibody, sandwich ELISA could be divided into three forms, direct sandwich ELISA, indirect sandwich ELISA, and sandwich ELISA with streptavidin-biotin detection.

Sandwich ELISA with direct detection: the detection antibody is enzyme-conjugated.
Indirect sandwich ELISA: the primary antibody used is unlabeled, and a secondary enzyme-conjugated detection antibody is required.
Sandwich ELISA with Streptavidin-Biotin Detection: the detection antibody used is biotin-labeled, and the extra streptavidin-HRP is required for signal amplification.