I am doing some IHC experiments and I have some problems while staining gut biopsies. There is faded brown shade on epithelium and tubular glands around Goblet cell. Now I need to know if this is an expression pattern of the particular protein or my mAbs have failed to maintain specificity. The Abs I am using are mAbs. Specific for epitopes.
Now one theory could be binding of similar epitopes, but how to configure if it's non-specific?
About 50% of all monoclonal antibodies are anything but specific. It was found for self made antibodies as well as in commercial antibodies (Kießig, ST, Jahn, S, Hiepe, F, Volk, H-D, Fietze, E, Zugehör, M, Porstmann, T, v.Baehr, R (1990) Multireactive human monoclonal antibodies. Allerg Immunol 36:163-177). So you have check the antibodies in your system of interest. In immunhistological setup you have the presence of an antigen cocktail present. The antigen which is either in the highest concentration present in the tissue section and/or the antigen recognized with the highest affinity. Therefore you can imagine how complex an experimental setup could be.
But,
1. you need to check the monoclonals in context of your question. The first part is to define the specificity of you antibody. Compare different methods. Simple and easy: competition with free (different concentrations) antigen (as pure as possible, e.i. peptide), also simple antibody absorption (with different pure antigen concentration at an solid phase e.i. latex particles), compare to ELISA (also using the competition experiments), western blot, ...
2. check the potential cross-reactions (which can disturb your assay, all others are not interesting)
3. Compare your results made with different antibodies. The major issue here is that several companies selling the same antibody with different names, but the original clone is the same.
4. If available, use polyclonals. Here you can expect crossreactions but no multireactivity. Here are so many antibodies present (up to 10E8 different), that the individual multireactivity of an single clone (of the 10E8) will not be detectable more than a little background.
5. For the second antibody choose one, which is really species specific (http://www.seramun.com/products/antibodies/labelled.html).
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