Isotype Controls

What is an antibody isotype?

An isotype is a broad class of antibody, determined by the structure and conformation of the heavy chain in the constant (Fc) region of the antibody. There are five types of mammalian heavy chain, referred to as alpha, delta, epsilon, gamma and mu. These define the five antibody isotypes: IgA, IgD, IgE, IgG and IgM, respectively. Further subclassifications exist for IgA (IgA1-IgA2) and IgG (IgG1-IgG4) in mammals, and substantial variation exists between species, for example rabbits have 14 IgA subclasses and lack IgD, while chickens have IgA, IgY and IgM isotypes.

Isotype structure determines the valency of the antibody, referring to how many antigens the antibody can bind at once (Figure 1). IgG has the classic Y-shaped antibody structure, with the antigen-binding (Fab) variable regions able to bind to two antigens in total. IgD and IgE have similar Y-shaped structures, albeit with different hinge region structures, and so also have a valency of two. In contrast, the dimer form of IgA has a valency of four, and IgM often exists as a pentamer with a valency of 10.

What are the functions of antibody isotypes?

In the immune system, antibody isotypes have different functions and are prominent at different stages of the immune response.

IgG

IgG is the most abundant antibody in the blood of mammals, representing 70-85% of the total Ig pool, though it can also be found in tissues. It is the primary component of the secondary immune response, which occurs when the immune system is exposed to an antigen that it has already encountered at least once. The long half-life of IgG means that it can mediate immunity to pathogens for months or years.

IgG has several important functions, including:

• Binding to and immobilizing pathogens and toxins
• Opsonization, enhancing phagocytosis by phagocytes such as macrophages
• Activating the classical pathway of the complement system
• Mediating antibody-dependent cell-mediated cytotoxicity (ADCC)

IgM

IgM represents 5-10% of the total Ig pool. It is important for the primary immune response that occurs when encountering an antigen for the first time, and can also activate the classical pathway of the complement system by binding C1. Its large, pentameric structure means that it tends to be restricted to the blood.

IgA

IgA accounts for 5-15% of the total Ig pool and is present as both monomer and dimer in mucosal secretions such as saliva, tears, milk and mucus, from which it protects mucosal membranes.

IgE

IgE is synthesized within plasma B cells and interacts with basophils and mast cells, with very little IgE found in circulation. Though IgE is thought to be involved in the immune response to parasites, it is best known for its role in hypersensitivity to allergens, such as in asthma, sinusitis, food allergies, and anaphylaxis in response to drugs.

IgD

IgD is only approximately 1% of the total Ig pool, typically found on the surface of B-lymphocytes alongside IgM. IgD is the least well characterized of the mammalian isotypes, but it is highly evolutionarily conserved and appears to be a marker of B cell activation

IgY

IgY is the major antibody isotype in birds and reptile serum as well as egg yolk. It is analogous to the mammalian IgG and performs similar functions, though it does not activate the complement system. It is structurally distinct from IgG and is not recognized by antibodies raised against IgG.

What is an isotype control?

During the antibody staining procedure, it is important to note that the primary antibody can bind not only to its target antigen but also to non-specific molecules such as reactive epitopes or Fc receptors present on cell surfaces. This non-specific antibody interaction (off-target interaction) can produce a background signal that may confound experimental results and interpretation. It is therefore important to distinguish between the specific signal and the background signal produced by antibody binding. This is achieved by using an isotype control antibody, which is an antibody raised against an antigen that is not expressed in the analyzed sample. The isotype control differs from the primary antibody only in the specificity for the antigen.

Why are isotype controls important?

Isotype controls are negative controls that help to ensure the specificity of the primary antibody staining. Isotype controls are primary antibodies that do not bind to any antigen in the sample but match all the properties of the experimental primary antibody, such as: host species, isotype, and conjugation. This is particularly relevant for techniques in which there is a high risk of background staining, such as in flow cytometry and IHC where endogenous Fc receptors are present. Because it is not possible to predict the unspecific binding of a primary antibody to non-target molecules, an isotype control is necessary to evaluate the contribution of non-specific background signal to the staining results.

What causes non-specific background staining?

Background signal occurs when primary antibodies bind non-specifically to the Fc receptors expressed on cells, such as B cells, macrophages, monocytes, and dendritic cells. The primary antibody can also bind non-specifically to lipids and carbohydrates and can interact with off-target proteins or debris present in the sample. Isotype controls are able to control for any off-target interactions that occur with the Fc region of the antibody, which is consistent within an isotype class. Off-target binding effected by the Fab region, such as if a target epitope occurs in multiple proteins, would not be picked up.

How to choose an isotype control?

An isotype control antibody must match the properties of the primary antibody being used. It must be raised in the same host species, be of the same isotype and Ig subclass, and have the same conjugation. For example, if you are using a PE-conjugated mouse IgG1 monoclonal antibody, you should use a PE-conjugated mouse IgG1 isotype control.

Which experimental conditions should you consider?

The isotype control antibody should be used in place of the primary antibody, but otherwise all other experimental conditions should be kept constant, including: working concentration (μg/ml), temperature, incubation, and detection method (e.g. use the same secondary antibody). The isotype control itself should be as similar as possible to the experimental antibody. As well as host, isotype and conjugation, this includes the clonality, its purity, and even the supplier providing the antibody. The supplier is particularly important for conjugated antibodies because the isotype control should have an equivalent label-to-antibody ratio as the primary antibody, and conjugation methods and the resulting number of fluorophore molecules conjugated to a particular antibody can vary between suppliers.

How can you reduce non-specific antibody binding?

Once non-specific binding has been identified through the use of an isotype control, it can be helpful to try to block as much of the non-specific binding as possible in case it is masking genuine signal. Non-specific antibody binding can be reduced by blocking Fc receptors, for example by adding an excess of BSA to the buffer. Additionally, reducing the amount of antibody being used, performing a titration to find the optimal primary antibody concentration, and gating out dead cells in flow cytometry experiments by using live / dead markers can all help to reduce non-specific signal.

Key Points

• An isotype control is an antibody that possesses equivalent properties to the primary antibody (i.e. host species, Ig subclass, and conjugate) but does not recognize any antigen in the sample.
• The isotype control should be used as a negative control in place of your primary antibody.
• An isotype control is important in order to evaluate the specificity of the primary antibody and to enable you to differentiate between specific antibody staining and non-specific background signal in your experiments.
• An isotype control works well when the primary antibody staining is easily distinguished from the non-specific background signal obtained from the isotype control. Therefore, the isotype control antibody should not recognize any specific antigen in the sample.
• Isotype controls are commonly used in flow cytometry and immunohistochemistry as these applications are likely to have high background signals.

Recommended Isotype Controls