Competitive ELISA Explained For Beginners

ELISA is an abbreviation of enzyme linked immunosorbent assay and utilize the bond between an antibody and its specific antigen for it to work. There are 4 main types of ELISA:

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA

Today we will look closer at competitive ELISA which is carried out in the following manner:

1. First, the sample is mixed with an excess of antibodies specific to the target antigen. These antibodies react with the antigen and form an antigen-antibody complex.
2. Second, the mixture is added to a micro well plate, with wells containing antigens that also are specific to the antibodies that were mixed in with the sample. However, only unbound antibodies can bind to the antigens that the plate is coated with since in the antigen-antibody complex, the antibody has already bonded.
3. Third, enzyme-conjugated secondary antibodies which bind to the first set of antibodies are added.
4. Fourth and finally, a substrate specific for the enzyme which is linked to the secondary antibody is added, and the enzyme converts this substrate into an observable signal.

This means that the more target antigen is present in the sample, the more antibodies are bound by it. As a result, the less free antibodies are left to bind to the antigens coated to the wells. This results in that less enzyme conjugated secondary antibodies can bind and produce a signal, hence resulting in a weaker signal. In other words the more of a color change occurs, the LESS target analyte is present in the sample.

It is also important to note that the way competitive assays are carried out may vary slightly but they all follow a similar structure as the one laid out here. However, sometimes, labeled antigens same as the target antigen are mixed with the sample. In this case the plate has been coated with capture antibodies specific to these and as such, they have to compete for binding spots. The essential idea is still the same and the signal is also in this case inverse.

[LucasLearnz]

Please ask if anything was still unclear to you! Most likely more people than you are wondering the same thing!! They are just too chicken to ask!😉

[shukoor]

Thank you so much man, I had never properly understood competitive elisa

[Sp-qkdw]

Nice video, but why the heck do you draw the antibody with its binding to the antigen on its FC region and not on its FAB regions, its very misleading imo?

[bhavyamsinha]

Amazingly explained each type of ELISA!

[jojopompador]

how is the antigen-antibody complex first made not recognized by the secondary antibodies ? if im assuming correctly they shouldnt be able to recognize them because we only want the antigen-antibody complexes made by the unbound antibodies correct??

[FaithDaniel-pkzg]

this is what I will write for my exams!!!!