Four Types of ELISA-CUSABIO
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Thirdly, the secondary detection antibody binds to the primary detection antibody, and then the enzyme reacts with its substrate to produce a visible signal that can be measured.
In fact, each of the three formats, direct, indirect, and sandwich, can be adapted to the competitive format. In competitive ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary antibody. Firstly, the unlabeled primary antibody is incubated with the sample containing the antigen of interest, leading to the formation of antigen-antibody complex Ag-Ab.
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In this step, the antibody is excessive compared with the antigen, so there are free antibodies left. Secondly, the Ag-Ab mixture is added to the plate coated with inhibitor antigen that can also bind to the primary antibody.
The free primary antibody in the mixture binds to the inhibitor antigen on the plate, while the Ag-Ab complexes in the mixture do not and are therefore washed off.
Thirdly, the enzyme-labeled secondary antibody is added to the plate and binds to the primary antibody bound to the inhibitor antigen on the plate. Finally, a substrate is added to react with the enzyme and emit a visible signal for detection.
Through this procedure, you may find that the final signal is inversely associated with the amount of the antigen of interest in the sample, meaning that the more antigen in the sample, the weaker the final signal.
This is because primary antibodies bound to sample antigen will be washed off, while free primary antibodies left will be captured by inhibitor antigen immobilized to the plate and be measured by an enzymatic reaction.
Competitive ELISA described here is based on antibody capture, in which the plate is coated with antigen. There is another type of competitive ELISA that is based on antigen capture, in which the plate is coated with unlabeled antibody. Furthermore, competitive ELISA generally uses a labeled antibody for detection, but sometimes it uses labeled antigen instead of a labeled antibody.