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Shark-Whisperer t1_jb5i3up wrote

Signal amplification. The primary antibody is relatively unencumbered of detection tags, so can maximally bind it's target unimpeded. The secondary, often polyclonal, can bind multiple sites on the primary Ig antibody that's already attached to the target molecule. So multiple molecules of secondary (labeled) can attach to each primary antibody, thereby increasing signal strength, whether fluorescence, chemiluminiscence or colorimetric, versus using a labeled primary antibody alone.

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DurianBig3503 t1_jb5qbjp wrote

Moreover, price. If you want a new target you dont have to make a whole batch of new signalling AB just the primary AB of the same origin as before so they too can be targeted by the secondary AB.

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Aggressive-Apple t1_jb8lzbd wrote

This is the main reason. There are tens of thousands of antibodies for different epitopes available on the market, and hundreds of dyes, enzymes etc available for detection. Making all combinations would be excessively expensive. Instead it becomes a modular system, where you can choose your primary antibody depending on epitope, and your secondary depending on how you wish to detect it.

You can stain different things in different colors at the same time by using primary antibodies from different species, effectively creating orthogonal "channels".

In some cases, however, the primary-secondary method is inappropriate. For example when doing superresolution microscopy the two antibodies on top of each other can displace the dye too far from the molecule of interest. Then you may need a conjugated primary antibody, or a even smaller single-domain antibody ("nanobody").

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ursoda OP t1_jb5ilk0 wrote

Okay yeah that makes sense thank you!

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