which of the following ELLSAS
to different antibodies
Answers
Answer:
Explanation:
Direct ELISA
In direct ELISA, only an enzyme-labeled primary antibody is used, meaning that secondary antibodies are not needed. The enzyme-labeled primary antibody "directly" binds to the target (antigen) that is immobilized to the plate (solid surface). Next, the enzyme linked to the primary antibody reacts with its substrate to produce a visible signal that can be measured. In this way, the antigen of interest is detected.
Indirect ELISA
In indirect ELISA, both a primary antibody and a secondary antibody are used. But in this case, the primary antibody is not labeled with an enzyme. Instead, the secondary antibody is labeled with an enzyme.
The primary antibody binds to the antigen immobilized to the plate, and then the enzyme-labeled secondary antibody binds to the primary antibody. Finally, the enzyme linked to the secondary antibody reacts with its substrate to produce a visible signal that can be measured.
Sandwich ELISA
In direct and indirect ELISA, it is the antigen that is immobilized to the plate. In sandwich ELISA, however, it is the antibody that is immobilized to the plate, and this antibody is called capture antibody. In addition to capture antibody, sandwich ELISA also involves the use of detection antibodies, which generally include the unlabeled primary detection antibody and the enzyme-labeled secondary detection antibody.
Firstly, the antigen of interest binds to the capture antibody immobilized to the plate. Secondly, the primary detection antibody binds to the antigen. 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.
More details about direct ELISA protocol, indirect ELISA protocol, and sandwich ELISA protocol, please check it here.
Competitive ELISA
Compared with the three ELISA types above, competitive ELISA is relatively complex because it involves the use of inhibitor antigen, so competitive ELISA is also known as inhibition ELISA. 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. Here is a procedure of competitive ELISA:
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). 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.
Comparison of direct, indirect, sandwich, and competitive ELISA
Now we know how the four most common types of ELISA work, but how to choose the right type for your experiment? To find the answer, you need to understand the advantages and disadvantages of each ELISA type.
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