What are isotype controls? 

Immunoglobulin Classes

Figure 1. The five immunoglobulin classes or isotypes of immunoglobulins

An isotype control is an antibody selected as a negative control to detect non-specific binding in antibody-based experiments.

Understanding antibody structure is important for understanding how to choose an isotype control. Antibodies (immunoglobulins, Ig) are composed of two heavy chains and two light chains. In humans, there are five heavy chain isotypes α,δ,γ,ε,μ, corresponding to five antibody isotypes: α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM).

In addition to the heavy chains, an antibody has one of two light chain isotypes, κ and λ, that may also play a role in the physicochemical properties of antibodies (Figure 1; references 1, 2).

A monoclonal isotype control is a negative control antibody from the same species, immunoglobulin class, subclass, and light chain as the primary antibody used in a particular scientific application.

Isotype control antibodies are developed in a similar way to primary monoclonal antibodies. Unlike primary antibodies, the immunogens are unrelated to like-proteins or epitopes present in the target organism or species. This avoids unwanted antibody-isotype control antigen interactions. It is important that the isotype and conjugate (e.g. FITC, PE, Biotin, Unconjugated) of the control matches the isotype and conjugate of the primary antibody exactly. The isotype of a particular antibody is usually specified by the supplier.

Why is it important to use isotype controls?

Primary monoclonal antibodies can interact both specifically and non-specifically with Fc receptors on cells, blood proteins, cellular proteins, carbohydrates, lipids, and tissues, which can lead to non-specific binding and misleading experimental results.

The appropriate isotype control antibody is therefore used to ensure the generation of reliable data in many applications, including:

  • In vivo: pre-clinical animal studies requiring functional antibodies
  • In vitro: assays such as flow cytometry, immunohistochemistry, fluorescent immunocytochemistry, western blotting, ELISA, and Luminex® Multiple Assays

The focus here is on in vivo applications.

How to Select Isotype Controls for in vivo Studies 

There are a few important points that should be considered to ensure that isotype controls can differentiate non-specific signals from a positive signal, ensuring that the experimental result is generated by the primary monoclonal antibody and is not a false positive or false negative:

  • Match the isotype control antibody to the same host species, class, and subclass (e.g., hIgG1, hIgG2, hIgG3, or hIgG4) as the primary antibody being used (Table 1). It is also desirable to match the light chain, which may affect the physicochemical properties of antibodies (1, 2).
  • Isotype controls and primary antibodies should be of in vivo functional grade, selected based on purity specifications such as low endotoxin level, low presence of aggregates, no detectable leached protein A, and other contaminants that might cause false negatives or positives during preclinical studies.

Figure 2. Purity and reproducibility of Leinco Isotype controls. A, Overlay of chromatograms representing three different lots of a Leinco isotype control. B, Two separate lots of Human IgG1 Isotype Control (Clone MOPC21.1, Lanes 2 and 3), further illustrating the purity and reproducibility of Leinco antibodies.

Examples of in vivo Functional Grade Primary An