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Why is the secondary antibody conjugated to the enzyme in ELISA, instead of the primary antibody? Wouldn't it be easier to conjugate the enzyme to the primary antibody?

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1 Answer 1

up vote 4 down vote accepted

Making an antibody-enzyme conjugate isn't trivial. By using a primary/secondary set-up you can use the same well-characterised conjugate in combination with many different primary antibodies (as long as these primaries are all raised in the same species). There is also the possibility of some amplification: for example, if the secondary is an anti-Fab then two secondary Igs will bind to each primary.

Response to OP comment

Most primary antibodies in common use are derived from rabbit or mouse, and most are IgG. So, for example, the secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG will cover most experiments.

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In other words; otherwise you would have to make a conjugate for every primary antibody? –  JohnPhteven Dec 15 '12 at 21:04
Yes, that's right. –  Alan Boyd Dec 15 '12 at 21:54
@ZafarS: in fact, that is what you do for most immunological techniques, like Western blotting, immunohistochemistry etc.: use an unconjugated primary and a conjugated secondary. –  nico Dec 16 '12 at 10:20
@AlanBoyd Sorry, I still have some doubt whether I grasp secundary antibodies. Every species has (or better: can have) a secondary antibody per animal right? So for example; we can have secondary antibodies for primary antibodies of rabbits, chimps, mice, etc. So that would still be a lot of possible secondary antibodies, but anyway less than primary antibodies. –  JohnPhteven Dec 17 '12 at 21:13

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