Submitted by ursoda t3_11k1x13 in askscience
I learned this before but totally forgot and now I'm curious.
Usually, a secondary antibody that contains the tag is used to recognize the primary antibody which binds to the antigen of interest.
There's a reason why the primary antibody isn't tagged usually, but what is it? Does it affect the sensitivity of the assay?
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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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Okay yeah that makes sense thank you!
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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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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Thanks for this - in the future, please report the comment, it's faster for us to get to.
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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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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").
Thank you for the advice, will do!
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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[deleted] t1_jb5f01v wrote
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