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How to select a secondary antibody?

Rosaria Esposito
Tags: Successful Research Tips

Antibodies (Ab), also known as immunoglobulins (Ig), are large glycoproteins produced by the immune system to bind and neutralize a pathogen or a foreign substance. The capability of antibodies to specifically recognize a unique target has been exploited in a variety of fundamental laboratory methods. One of the most typical applications is their use as a targeted arrow for the detection of a molecule of interest, with techniques such as western blot, ELISA, electron microscopy, flow cytometry or immunostaining (i.e. immunohistochemistry, immunofluorescence, immunocytochemistry). The antibody directly binding the antigen is called primary antibody (AbI). In direct detection methods, the primary antibody is, itself, conjugated to a reporter, such as a fluorophore, or an enzyme able to convert a substrate into a visible product. However, the preferred strategy is often an indirect detection, where the unlabeled primary antibody is recognized by a labeled secondary antibody (AbII). Since several secondary antibodies can bind different epitopes of the primary antibody, indirect method ensures an amplified signal and a higher sensitivity. In addition, it also allows higher flexibility as just few types of secondary antibodies can be used to detect a wide range of primary antibodies against the different antigens. If the choice of the AbI is pivotal, the selection of the AbII is also extremely important to obtain the best possible result. A list of tips for the selection of the most suitable secondary antibody for your experiment is presented below.

  1. What is the species of the primary antibody?

    The secondary antibody has to be raised against immunoglobulins from the host species of the primary antibody. This means that if, for example, the primary antibody has been generated in mouse, the secondary antibody needs be directed against mouse immunoglobulins. In addition, it should not derive itself from mouse, as the host species for the primary and the secondary antibody should be different. Most common primary antibodies are generated in mouse, rat, rabbit, hamster or goat, thus a selection of secondary antibodies against these species and from different sources is commercially available.


  2. What are the class and subclass of primary antibodies?

    Based on the heavy chain they contain, antibodies can be classified in five classes or isotypes: IgG (the most abundant in serum), IgA, IgD, IgE, and IgM (Figure 1A and 1B). In addition, IgG and IgA are further divided in subclasses (e.g. IgA1, IgA2, IgG1, IgG2, etc). Therefore, secondary antibody needs to match not only with the species but also with the isotype/class of the primary antibody. Polyclonal antibodies generally belong to the IgG class. The subclass is relevant especially for monoclonal antibodies, as each of them belongs to a specific one. The possibility of selecting primary antibodies of different classes/subclasses can be exploited for instance in multiplexing (e.g. two mouse primary antibodies belonging to different isotypes/subclasses could be used together if isotype-specific secondary antibodies are available).


  3. What purity degree do you need?

    Antibodies can be purified by affinity chromatography. Proteins A or G bind the Fc-region of IgGs and can therefore be used as affinity ligands on the chromatographic columns to isolate and eventually elute the antibodies. If a higher degree of specificity is required, the antibody target antigen itself can be immobilized on the column, thus allowing the purification of all the immunoglobulins present in the preparation, based on their specificity for the antigen, regardless of their class. The purification method is usually mentioned in the technical datasheet of the commercial antibodies in order to help the final user in choosing the most appropriate one for the specific experimental conditions.


  4. Have you considered to pre-absorb the secondary antibody?

    Pre-adsorption (or cross-adsorption) is useful to reduce possible background issues, especially when multiple primary antibodies from different species are used simultaneously. With this method, antibodies from species other than the target one are immobilized on a column. The medium containing the secondary antibody passes through this column and only the non-cross-reacting components will flow through. Potential undesired cross-reactivity with immunoglobulins from non-target species are therefore eliminated. For example, if a mouse primary antibody is used, a secondary antibody cross-adsorbed against rat, human, rabbit, and goat could lead to a cleaner result.


  5. Have you considered using antibody fragments?

    It is possible to detect the primary antibody just using the variable region of the secondary one, (Figure 1C). The bivalent variable region, known as F(ab’)2, is obtained by pepsin digestion of IgG antibodies, whereas a monovalent fragment, F(ab’), derives from papain digestion. Thanks to their smaller size, antibodies fragments can penetrate tissues more easily than the whole immunoglobulin. Furthermore, they are particularly useful to avoid cross-interaction with cells typically exposing Fc receptors, such as NK cells, leukocytes, etc.


  6. Schematic view of an IgG

    Schematic view of four Ig classes

    Antibodies fragments

    Figure 1: Immunoglobulins representation. A. Schematic view of an IgG. B. Schematic view of the other four Ig classes. C. Antibodies fragments F(ab’) or F(ab’)2 obtained after papain or pepsin digestion.
  7. What is the reporter you need?

    This aspect is strictly related to the desired application. Secondary antibodies are, for example, usually conjugated with reporter enzymes (Alkaline Phosphatase (AP) or the Horseradish Peroxidase (HRP)) for western blot, ELISA or chromogenic immunohistochemistry; fluorophores are instead often for immunofluorescence or flow cytometry.


Enzo Life Sciences catalogue offers a selection of typical secondary antibodies, conjugated with the most common reporters (e.g. HRP, AP, R-PE, ATTO 590). You can browse them following this link: Secondary Antibodies. If you are looking for solutions to increase the sensitivity, the specificity, and the signal intensity of the IHC experiments do not miss our POLYVIEW PLUS/MULTIVIEW PLUS nanopolymer based detection solutions. Check out our successful research tips including our Top 10 Tips for Choosing an Antibody. Finally, do not hesitate to get in contact with us to find out all about our custom conjugation services with different reporters. For all questions and concerns, our Technical Support Team is here to assist.

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