IHC: How to Select a Primary Antibody and Optimize It

Immunohistochemistry must be performed using specific antibodies which bind to the epitope that is being studied. This article discusses this in brief, as well as other factors which help to determine which primary antibody to select.

Specificity for the Target Epitope

In most cases, the specific binding of the antibody to the protein must be found by experimental means. Alignment tests, such as BLAST, can offer a comparison of the immunogenic sequence of the antigenic protein and other proteins, to suggest the presence of specificity. However, its results are not definitive.

The best and most reliable way to determine the specificity of an antibody is detecting the absence of staining in tissues or cells after the target protein (to which the antibody binds) has been knocked out. Other tests include recognizing a single band on a western blot test, though this is not too dependable either; or finding staining in patterns that fit existing knowledge of where the target protein is localized in control tissues or cells.

In the ideal case, the antibody should be able to recognize the target antigen in the species being studied. If not, it may be necessary to compare the immunogen sequence with the region that corresponds to it in the protein obtained from the species being studied, to detect specificity to some extent.

Does the Antibody Have a Track Record with IHC?

Even if an antibody does bind to the target epitope or antigen during a western blot, it is important to understand that this takes place in a situation where the antibody is denatured. In such an experiment as IHC, where the antigen exists in its native 3D form, this recognition may fail to occur.

Thus, it is vital to choose an antibody that has already been shown to work in an IHC or an immunocytochemistry (ICC) experiment. The antigen-antibody binding is also affected by whether the antibody can recognize the epitope being studied after fixation and antigen retrieval have been performed by various methods.

Is the Antibody Monoclonal or Polyclonal?

Monoclonal antibodies are those that originate from just one B cell clone. These are therefore identical, forming a population of homogeneous molecules, which recognize and bind to the same single epitope.

This narrows the likelihood of cross-reaction between the antibody and the antigenic site of other proteins as long as the antibodies are specific to the epitope they recognize. This results in low background staining. Moreover, they are produced by a special cell called a hybridoma which leads to the production of antibody batches with little inter-batch variation.

Polyclonal antibodies, on the other hand, form a heterogeneous group of molecules which are designed to recognize and bind to more than one antigenic site. They can therefore be of use when a protein has undergone some change in its conformation due to fixation, for instance, or heating/cooling, helping to increase the likelihood that the antibody will still recognize it. They also have improved pH stability, and tolerate changes in the salt concentration more easily than monoclonal antibodies do.

Which is the Host Species?

The primary antibody should be raised, under ideal conditions, in a species that is not the same as the one from which the sample will be taken, so that the host’s own immunoglobulin or antibody molecules will not cross-react with it.

Optimizing the Antibody

How good a staining is depends not only on the concentration of the primary antibody, but also the kind of diluent used, the time allowed for incubation and the temperature. Each must be individually adjusted to optimal levels for each of the samples and each of the antibodies, so that the staining will result in specific patterns and the least possible background staining.

This is usually done by varying the concentration of the antibody but keeping the time allotted for incubation as well as the temperature unchanged. If the tissue is incubated longer, there is more surety that the antibody will penetrate the tissue adequately. If this is performed at cooler temperatures as well as for a longer time of incubation, specific binding is more likely to occur.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.

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Last updated: Jan 24, 2019 at 11:21 AM


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