​Isotype Controls: An Important Experimental Tool

​Isotype Controls: An Important Experimental Tool

25th Oct 2023

When conducting immunology experiments that utilize antibodies, it is critical to include the proper controls. One such control is the isotype control, which can help determine whether results are due to specific antibody binding or non-specific background signal. Here we explain what isotype controls are and why they are an essential part of many experiments.

Table of Contents

  • What are antibody isotypes?
  • What is an isotype control?
  • Do I need to use isotype controls?
  • How to choose an isotype control
  • Analyzing isotype control results
  • Uses of isotype controls in vivo
  • Isotype controls in human studies
  • Causes of background staining
  • What are antibody isotypes?

    Antibodies (also known as immunoglobulins) are composed of two types of protein chains – heavy and light (Figure 1). 

    Figure 1: Typical immunoglobulin structure showing heavy and light chains.

    In mammals, heavy chains comprise five different classes, or isotypes: IgM, IgA, IgD, IgG and IgE. These isotypes differ in their biological functions and physiological locations, and are adapted to deal with different antigens during an immunological response.

    The five heavy chain isotypes are denoted by Greek letters: μ (IgM), α (IgA), δ (IgD), γ (IgG), and ε (IgE). Of these, IgG is the most abundant and consists of several subgroups (Figure 2).

    Figure 2: Schematic representation of all antibody isotypes in mammals

    What is an isotype control?

    An isotype control is an antibody that lacks ability to bind the target antigen, but matches other properties of the primary antibody. Isotype controls are used as negative controls in place of primary antibodies to determine contribution of non-specific background staining, and distinguish specific and non-specific antibody staining.

    Isotype controls are often used in flow cytometry and immunohistochemistry where background staining is common. However, they may be used in any assay where background interferes with results. They can also be blocking reagents in immunoassays like western blots and ELISAs.

    Do I need to use isotype controls?

    The use of proper controls, including isotype controls, is imperative for rigorous experimental design and reliable data interpretation in antibody-based assays. Isotype controls serve as essential negative controls by accounting for non-specific background signal, distinguishing specific from non-specific antibody binding. While the necessity depends on factors like assay type and target sample, isotype controls provide key insights into background staining levels. Their inclusion strengthens the specificity of results by enabling discernment of true positive signal. Thus, incorporating isotype controls is recommended to support robust experimental conclusions.

    How to choose an isotype control

    Selecting an optimal isotype control involves matching key antibody characteristics including host species, isotype subclass, and conjugation to the primary antibody. Additionally, utilize the same experimental conditions between the paired antibodies like concentration, incubation parameters, blocking solutions, and detection methods for valid comparisons. Matching these critical properties while minimizing procedural differences allows the isotype control to act as an accurate negative surrogate for interpreting specific binding. With careful antibody characterization and controlled testing conditions, the isotype control reliably models background noise, enabling specificity determinations.

    Analyzing Isotype Control Results

    Compare signal from primary antibody to isotype control run under same conditions. Minimal staining indicates low background. Considerable isotype control signal reveals background level to interpret actual antibody binding signal.

    While isotype controls reveal background staining, they don't confirm antibody specificity or indicate background source. Still, they are an essential control for reliable immunology experiments.

    In summary, isotype controls are an essential experimental tool for immunology studies. They help control for non-specific background signal and antibody effects like HAMA interference. Careful selection of the appropriate isotype control for each target antibody and sample type allows you to confidently interpret the specific binding in your experiments. Isotype controls provide an additional level of scientific rigor that produces reliable immunology data.

    Uses of isotype controls in vivo

    When performing in vivo experiments, isotype controls are extremely useful for analyzing cells and tissues by flow cytometry or microscopy. Tissues contain many proteins that antibodies can bind to non-specifically. Using an isotype control antibody helps determine the level of background signal in your experiment. Samples labeled with the isotype control can then be compared to those labeled with the specific antibody. Any signal greater than the isotype control is likely due to specific binding of the target antibody.

    Isotype controls for human studies

    For experiments involving human samples, choosing the right isotype control is critical. Since the test antibody and isotype control are both mouse antibodies, they could cause false positive signals by binding to human anti-mouse antibodies (HAMA) present in patient samples. Using a mouse antibody with the same isotype but different specificity helps control for these effects. An IgG1 control is standard for human IgG1 antibodies, but for other subtypes like IgG2 or IgM, an antigen-specificity matched control provides even better specificity. With the right isotype-matched controls, you can effectively identify specific target antibody binding in human samples.

    Causes of background staining

    Although tissue-dependent, background staining can be attributed to:

    • Binding to Fc receptors on target cells
    • Cellular autofluorescence
    • Non-specific antibody interactions

    Mouse antibodies like IgG2a may bind Fc receptors on some human leukocytes, requiring an IgG2a isotype control.