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From a short introduction to immunohistochemistry controls:

Isotype Control

This control can be utilized when working with monoclonal primary antibodies. The sample is incubated with antibody diluent, supplemented with a non-immune immunoglobulin of the same isotype (for example, IgG1, IgG2A, IgG2B, IgM) and concentration as the primary monoclonal antibody. The sample is then incubated with the secondary antibody and detection reagents. These steps will help ensure that what appears to be specific staining was not caused by non-specific interactions of immunoglobulin molecules with the sample. Background staining should be negligible and not resemble specific staining.

I googled and found out that some pathological agents can bind immunoglobulins in a non-immune fashion, i.e. by binding to their Fc regions. But that still leaves me in the dark on "non-immune IGs".

Maybe it is an IG with which the secondary antibody will not bind?

Or maybe it's an IG that has no activity against the target antigen?

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The term "non-immune immunoglobulin" is both confusing and non-standard in the immunology field - "pre-immune" is sometimes used, but not always accurate. A negative control is as they describe - the same diluents and components of the sample incubated with the antibody of interest, only they use a non-specific antibody of the same isotype that is not expected to bind to any targets in an "immune-dependent" manner - the "usual" way you think of an antibody binding to a target antigen. However, antibodies can bind samples in an immune-independent manner, via protein-protein interactions (the sample was insufficiently blocked) or to Fc receptors, for example.

The non-specific antibody can be from several sources - a pre-immunization bleed from the animal that generated a polyclonal primary antibody, an antibody to a target not expected to be present in the sample, such as KLH or GFP, or a monoclonal antibody engineered or screened to not bind to anything.

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In theory, "non-immune antibodies" are any kind of antibodies that do not bind the sample using their Fab, specific regions. See for example https://books.google.com/books?id=sFMJAwAAQBAJ&lpg=PA80&ots=O9CqXW9kYt&pg=PA80#v=onepage&f=false Here, a claim is made that they might still bind non-specifically using their Fc regions.

In practice, one ends up using a generic mixture of all immunoglobins from a species' blood (here, all "all immunoglobins of a specific isotype"). It is prohibitively expensive to deplete this mixture of specific antibodies for all the proteins in your sample. You could also imagine some denaturation that renders Fc regions inactive. I couldn't find evidence of either, so generic mixture it is. See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1162460/pdf/biochemj00424-0223.pdf for an example of "non-immune" preparation - it is simply a precipitation of all IgG. It would be great if you could prove the generic mixture has very little or no specific antibodies to your samples*, but I doubt anyone bothers.

Another option is a monoclonal Ab that works against a completely unrelated protein. Again, one should prove their is no specific reactivity* with the samples.

Pro-tip: searching for "non-immune IgG" will bring way more results than "non-immune immunoglobulin". In general, "immunoglobulin" is less used than the synonymous "antibody".

* This means cleaving the Fc regions, separating them, labeling them with radioactivity or fluorescence, and trying them on a sample. It's tedious and expensive.

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  • $\begingroup$ Check out my answer, it addresses some of the things you mention here. In practice, it's actually rather easy to make a non-specific negative control antibody. $\endgroup$ – MattDMo Aug 5 '16 at 18:14
  • $\begingroup$ If the "non-immune antibodies" are 100% not binding the sample, it means that their Fc is also guaranteed not to bind the sample. That makes their use as control in the protocol described in the question rather perfunctory. I am not sure what's worst as a control: a serum which may bind the sample (the way I said it) or a serum whose Fc is guaranteed not to bind (your variant). $\endgroup$ – Nick Alexander Aug 5 '16 at 18:24
  • $\begingroup$ I was talking about the Fab region, not the Fc. You definitely don't want to make an isotype control whose Fc doesn't bind to Fc receptors - that would completely defeat the purpose of using a control. What I was talking about was engineering or screening a clone with a Fab that doesn't bind any known antigen, but the rest of the scaffold is identical to your specific antibody of interest. That way, any binding/staining you see with it is truly non-specific (non Fab-mediated). $\endgroup$ – MattDMo Aug 5 '16 at 19:12

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