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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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Jordanno99 t1_jb6leae wrote

Often you are interested in a primary antibody that binds the antigen. Depending on where your primary antibody is from (serum, plasma, expression) it may not have a tag for direct detection, and it would be cumbersome to chemically add one.

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Shark-Whisperer t1_jb9wofh wrote

True, and a very good example is the multiplex kits available for simultaneously detecting multiple anti-SARS-CoV-19 isotypes (e.g., IgG, IgA, IgM) in blood in response to infection or immunization.

Antibodies are pretty stable molecules but physically attaching any additional moiety, such as a fluorophore, risks creating steric hindrance issues and reducing antibody binding to it's target antigen sequence. Even the smallest modifications such as biotinylation can slightly reduce binding in some instances.

These physical access issues can be more important with '2D' systems when the target is immobilized on flat microtiter plate bottoms, which effectively hides the bottom side of the molecule from exposure and target binding to the ELISA plate can alter its physical structure presented to the antibody, compared to 3D exposure of target/detection antibody in liquid/suspended environments.

Also, secondary antibodies are great for signal amplification, but non-specific background binding goes up, too, versus using a labeled primary ab. This isn't really an issue if appropriate controls are included, and the signal amplification is proportionally greater than any increased background binding.

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a-synuclein t1_jb5w8nw wrote

For ELISAs to show binding we used an HRP-conjugated protein that we conjugated with a kit (3 hour process, very easy to do) and it worked great. Plenty of labs conjugate the "primary" sensor (antibody or protein) and forgo secondary. There are disadvantages of course but sometimes you don't have a choice and it works fine with enough concentration.

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