ELISA immunoassays

[deleted-35387]

When researching about ELISA immunoassays, I understand that the protocol is basically this: We use an antibody specific against an antigen (the primary antibody) to detect the presence of an antigen. If the antigen is in the sample, then the primary antibody will bind. We then use a secondary antibody conjugated with an enzyme to detect the presence of the primary antibody. After rinsing, we add the substrate for the enzyme, and a reaction will occur depending on the presence of the secondary antibody, and because the presence of the secondary antibody depends on the presence of the primary antibody, which in turn depends on the presence of the antigen, this reaction will determine whether the antigen is present in our original sample. What I don't understand is, why couldn't we have just used a primary antibody conjugated with an enzyme? Why bother with a secondary antibody at all? Maybe there's some factor I'm missing...if someone could please explain this that would be very helpful!

Stephen

[Walker]

Hi Stephen,

You could indeed perform a basic ELISA using an enzyme-linked primary antibody. It's usually just easier to use a commercially-available enzyme conjugated secondary antibody (which are cheaply available and work for any ELISA using the same IgG species/isotype) than it is to make a custom-conjugated primary (which is a pain to do yourself, more expensive to purchase, and only works for that one ELISA test).

Say you have an antibody lying around the lab that you want to do an ELISA with. It's much simpler to spend $95 on an enzyme-conjugated "anti-rabbit IgG" secondary (which are readily available commercially) than it is to conjugate the enzyme to the primary yourself. And if you have, say, a dozen different rabbit antibodies that you want to do ELISAs with, you can use the same conjugated secondary for all of them, instead of having to produce a custom-conjugated primary for each reaction.

Using secondary antibodies also can give you a more sensitive assay, since more than one enzyme-labeled secondary can bind to each primary antibody. It's also possible to use tertiary reagents (such as a streptavidin/biotin ("ABC system")) to get even more signal amplification. You might find this discussion of ELISA helpful:

http://www.piercenet.com/Proteomics/bro ... 6FE09D8403

Best,
Will