Beginner’s Guide to Immunohistochemistry 2: Choosing a Secondary Antibody

blog / Pathology August 27 2019

Welcome back to our beginner’s guide to immunohistochemistry (IHC). In this post, we’ll follow on from our last article on choosing a primary antibody, bringing you the most important considerations for choosing a secondary antibody.


Match Host and Isotype of Primary Antibody

The secondary antibody should always target the species in which the primary antibody was raised. For instance, if you’re using a rabbit primary antibody, your secondary antibody should be anti-rabbit. You should also ensure that the isotype recognised by the secondary antibody matches that of the primary antibody.

Whole Immunoglobulins or Fab/ F(ab’)2 Fragments?

Consider whether you want to use whole immunoglobulin or antibody fragments. Fab and F(ab’)2 fragments are the antigen-binding regions of an antibody that remain when the Fc region is either wholly or partially removed. Enzymatic digestion of antibodies with either papain or pepsin results in Fab fragments that contain either one binding site and no Fc portion (monovalent antibodies) or F(ab’)2 fragments, which have two binding sites connected by a disulphide bonds (divalent antibodies).

Fab fragments are often used during IHC to block unspecific binding of conjugates to endogenous immunoglobulins in subsequent staining steps while F(ab’)2 fragments are often labeled and used as conjugates when staining tissues or body fluids that are rich in Fc receptors, such as the lymph nodes, thymus, and blood. Furthermore, because they are small, antibody fragments penetrate tissues more easily than whole immunoglobulins, making them a good choice in experiments that call for multiple IHC stainings.

Minimise Background with Pre-Adsorbed Antibodies

When hunting the web for antibodies for IHC, you will often come across pre-adsorbed secondary antibodies. Pre-adsorption is an extra purification step whereby an antibody solution is adsorbed i.e. exposed to immobilised animal or human serum to remove non-specific antibodies from the mixture. This increases the specificity of the adsorbed antibodies, thus reducing the risk of non-specific background staining.

While adsorption has its obvious advantages, adsorbed antibodies are often limited by markedly reduced epitope recognition and they may recognise certain IgG subclasses very weakly, in particular the subclasses that most closely resemble the species they were adsorbed against. Therefore, you should only use adsorbed antibodies if strictly necessary; for instance, you could consider using an anti-human IgG that has been adsorbed against mouse IgG if you plan to detect a human primary antibody in mouse tissue that contains mouse immunoglobulin.

Purification Level

Affinity purified antibodies are commonly used in IHC because they give minimal non-specific binding. However, IgG fractions may be useful in situations that call for high antibody affinity, e.g., when target antigens are scarce.

Labelling / Conjugated

Generally speaking, secondary antibodies are either enzyme-labelled e.g. with alkaline phosphatase or horseradish peroxidase (HRP), fluorochrome-labelled e.g., with FITC or Alexa-Fluor® or biotinylated. Alkaline phosphatase conjugates offer high sensitivity especially in colorimetric detection setups. Peroxidase is a stable enzyme that yields high intensity signals in short time periods and tends to be cheaper than other enzyme conjugates. Fluorochome-labelled or fluorescent antibodies are generally used in protocols that require multiple rounds of staining. Detection formats that use biotinylated antibodies exploit the strong affinity that exists between biotin and its binding partners, e.g., avidin and streptavidin. Biotinylated antibodies thus offer higher sensitivity than the aforementioned conjugate types because biotin-strept(avidin) complexes are very stable and resistant to fluctuating assay conditions, and each biotin molecule can bind multiple molecules of strept(avidin) thus amplifying the detection signal.

To achieve even higher sensitivity, for example when detecting very low-abundance proteins, HRP-polymer secondary antibodies are a good option. These antibodies use micropolymer technology to form smaller detection complexes that allow better tissue penetration and sensitivity. In addition, HRP-polymer secondary antibodies bind more horseradish peroxidase than standard HRP secondary antibodies, thus leading to higher intensity signals.

It’s Not Always Black and White!

In practice, the choice of secondary antibody comes down to the exact situation in hand; the level of sensitivity and purification required must be weighed up against the target abundance and the risk of non-specific binding, while the choice of labelling system may be partially influenced by the materials and equipment available in your laboratory. No matter what your setup is, you are always welcome to contact the team at Nordic BioSite, who will be delighted to assist you in finding the right antibody for your needs.