What is the difference between direct and sandwich ELISA?

The main difference between direct and sandwich ELISA is that direct ELISA uses only one antibody while sandwich ELISA requires the use of matched antibody pairs (capture and detection antibodies).

In direct ELISA, it is the antigen that is immobilized to plate, whereas in sandwich ELISA, it is the capture antibody that is immobilized to the plate. In addition to the capture antibody, sandwich ELISA also involves the use of detection antibody, which has a different and non-overlapping of region or epitope of the antigen from the capture antibody. The detection antibody can be enzyme labeled, in which case it is known as a direct sandwich ELISA. If the detection antibody is the unlabeled primary antibody, another enzyme-labeled secondary detection antibody is needed, forming an indirect sandwich configuration.

Sandwich ELISA generally has higher sensitivity and specificity than the direct ELISA because two antibodies are involved in capture and detection. Since antigen does not need to be purified prior to measurement, sandwich ELISA is a better choice for the analysis of complex samples than the direct ELISA. However, it is usually difficult to select and optimize the antibody pairs for sandwich ELISA and there might be cross-reactivity between the capture and detection antibodies.